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Full text of "Investigation of the kinetic mechanism of glutamine- and ammonia-dependent reactions of E. coli asparagine synthetase B using isotope partitioning and steady-state kinetics"

INVESTIGATION OF THE KINETIC MECHANISM OF GLUTAMINE- AND 

AMMONIA-DEPENDENT REACTIONS OF E. coli ASPARAGINE SYNTHETASE 

B USING ISOTOPE PARTITIONING AND STEADY-STATE KINETICS 



By 
POURAN HABIBZADEGAH-TARI 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1996 



ACKNOWLEDGMENTS 

I would like to thank the members of my committee, Dr 
cain, Dr Cohen, Dr Dunn, and Dr Richards for their guidance 
and suggestions. I would like to thank my advisor Dr Schuster 
for giving me the chance to do reseacrch in his laboratory. 
Although he was always busy with a lot of other things, the 
very limited time I had with him was filled with new ideas to 
solve problems. I would very much like to thank Dr Alison 
helping me with my research, and for being there for me 
always . 

I would like to thank all the friends I met in and out 
of Dr Schuster's laboratory for their support and 
encouragement . 

I thank my husband, Esfandiar, for being my friend for 
the past eighteen years, specially during this period of my 
life. I am grateful for his ongoing support and for lifting 
my spirit when it was down. 

I am specially grateful to my mom for coming to this 
country and taking care of my daughter while I was busy with 
school. I know how hard it was for her to stay in this 
country for ten months and not be able to communicate with 
anyone. I want thank her with all my heart and say how sorry 
I am for not being able to spend enough time with her. 



11 



Finally, I want thank my daughter, Maryam, for being who 
she is, my love. I hope I can make it up to her after I 
finish school. 






111 



TABLE OF CONTENTS 

page 

AKNOWLEDGEMENTS ii 

LIST OF TABLES vi 

LIST OF FIGURES vii 

ABSTRACT xii 

CHAPTERS 

1 INTRODUCTION 1 

2 INVESTIGATION OF THE MECHANISM OF E. COLI ASPARAGINE 
SYNTHETASE USING ISOTOPE PARTITIONING 26 

Introduction 2 6 

Materials and Methods 29 

Chemicals and Reagents 29 

Expression of the Protein and Purification 30 

Protein Concentration Determination 31 

Isotope Partitioning Experiments with 

Radioactive L-Aspartate, Glutamine- and 
Ammonia-Dependent Reactions 31 

Determination of Kd for L-Aspartate 34 

Isotope Partitioning Experiments with 

Radioactive ATP, Glutamine- and Ammonia- 
Dependent Reactions 34 

Aspartate-Dependent ATP Hydrolysis 3 6 

Theory 37 

Results 29 

Isotope Partitioning Experiments with 

Radioactive L-Aspartate, Glutamine- and 
Ammonia-Dependent Reactions 39 

Isotope Partitioning Experiments with 

Radioactive ATP, Glutamine- and Ammonia- 
Dependent Reactions 40 

Aspartate-Dependent ATP Hydrolysis '.'.'.'.'. 41 

Discussion "47 

3 SUBSTRATE BINDING AND PRODUCT RELEASE OF ASPARAGINE 

SYNTHETASE B STUDIED BY STEADY STATE KINETICS 55 

Introduction 'eg 

IV 



Materials and Methods 56 

Chemicals and Reagents 56 

Expression of the Protein and Purification 57 

Protein Concentration Determination 57 

Enzyme Assays 57 

Stoichiometry of PPi and L-Glutamate 59 

Results 60 

Initial Rate Studies 60 

Inhibition by Substrate Analogs 65 

Stoichiometry of Glutamine-Dependent Reaction 67 

Product Inhibition Studies 68 

Discussion 74 

4 EFFECT OF TEMPERATURE ON THE ASPARAGINE SYNTHETASE B....146 

Introduction 146 

Materials and Methods 146 

Chemicals and Reagents 146 

Expression of the Protein and Purification 147 

Protein Concentration Determination 147 

Enzyme Assays 147 

Thermodynamic of Activation 148 

Results and Discussion 149 

5 SUMMARY AND CONCLUSIONS 157 

LIST OF REFERENCES 166 

BIOGRAPHICAL SKETCH 172 



LIST OF TABLES 



Table page 

2 . 1 Trapping of L-aspartate from Complexes in the Steady- 
State, Using ( 14 C) L-aspartate, Ammonia- and 
Glutamine- Dependent Reactions 43 

2.2 Trapping of ATP from Complexes in the Steady State, 
Using ( 3 H) ATP, Ammonia- and Glutamine- Dependent 
Reactions 44 

3.1 Inhibition patterns for ASB obtained with ^-methyl 
aspartate, AMP-PNP and L-glutamic acid y-methyl ester 
with respect to L-aspartate, ATP and L-glutamine. .142 

3.2 Product inhibition data for ammonia-dependent 
reaction of ASB 143 

3.3 Product inhibition data for glutamine-dependent 
reaction of ASB 144 

4.1 Thermodynamic properties for ammonia- and glutamine- 
dependent reactions of ASB 156 



VI 



LIST OF FIGURES 



Figure page 

1.1 (a) Currently accepted mechanism for the hydrolysis 
of L-glutamine to yield ammonia and an acylenzyme 1 
by analogy with purF enzyme, GPA. (b) Synthesis of L- 
asparagine by reaction of ammonia with activated 
aspartyl derivative 2. (c) Hydrolysis reaction to 
yield L-glutamate from the acylenzyme 1 23 

1.2 Sequence alignment of the N- terminal domains of E. 
coli ASB and human AS as deduced from oligonucleotide 
sequencing 24 

1.3 Proposed mechanism for the synthesis of L-asparagine 
by E. coli ASB, via an imide intermediate 3 25 

2 . 1 Determination of Kd for Aspartate 45 

2.2 Rate of AMP formation as a function of time 46 

3 . 1A Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of ATP 96 

3 . IB Replots of the reciprocal-fixed variable substrate 
ATP vs slope (plus) and intercept (square) from 
Fig. 3 . 1A 97 

3.2A Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at various concentrations of 
ATP 98 

3.2B Replots of the reciprocal-fixed variable substrate 
ATP vs slope (plus) and intercept (cross) from 
Fig. 3 . 2 A 99 

3.3A Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-glutamine 100 

3.3B Replots of the reciprocal-fixed variable substrate L- 
glutamine vs slope (plus) and intercept (cross) from 
Fig. 3.3A 10 i 



VI 1 



3.4A Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of L- 
aspartate 102 

3.4B Replots of the reciprocal-fixed variable substrate L- 
aspartate vs slope (plus) and intercept (cross) from 
Fig. 3.4A 103 

3 . 5A Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of 
NH3 104 

3 . 5B Replots of the reciprocal-fixed variable substrate 
NH3 vs slope (plus) and intercept (cross) from Fig. 
3 . 5A 105 

3.6A Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of NH3 106 

3 . 6B Replots of the reciprocal-fixed variable substrate 
NH3 vs slope (plus) and intercept (cross) from Fig. 
3 • 6A 107 

3.7 Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at fixed concentration of 

LGH 108 

3.8 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed concentration of L-glutamine 
(plus) and LGH (cross) (0.2 mM) 109 

3.9 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of ATP HO 

3.10 Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at various concentrations of 
ATP Ill 

3.11 The ratio of L-glutamate produced/PPi produced versus 
concentration of L-glutamine 112 

3.12 Double-reciprocal plot of initial velocity versus NH3 
concentration at various fixed concentrations of L- 
asparagine 12.3 

3.13 Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of L- 
asparagine 2.14 



Vlll 



3.14 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-asparagine 115 

3.15 Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of 

AMP 116 

3.16 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of AMP 117 

3.17 Double-reciprocal plot of initial velocity versus NH3 
concentration at various fixed concentrations of 

AMP 118 

3.18 Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at various fixed 
concentrations of L-asparagine 119 

3.19 Double-reciprocal plot of initial velocity versus ATP 
at various fixed concentrations of L-asparagine. ..120 

3.20 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-asparagine 121 

3.21 Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of 

pp i 122 

3.22 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of PPi 123 

3.23 Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of L- 
glutamate _ 124 

3.24 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-glutamate 125 

3.25 Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at various fixed 
concentrations of L-glutamate 126 

3.26 Double-reciprocal plot of initial velocity versus L- 
glutamine concentration at various fixed 
concentrations of AMP 227 












ix 



3.27 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of AMP 128 

3.28 Double-reciprocal plot of initial velocity versus ATP 
concentration at various fixed concentrations of 

AMP 129 

3.29 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-glutamine in the presence of L- 
glutamate (50 mM) 130 

3.30 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at various fixed 
concentrations of L-glutamine in the presence of PPi 
(0.4 mM) 131 

3.31 Double inhibition studies plot of L-asparagine 
concentration versus reciprocal initial velocity at 
various fixed concentrations of AMP 132 

3.32 Double inhibition studies plot of L-glutamate 
concentration versus reciprocal initial velocity at 
various fixed concentrations of AMP 133 

3.33 Double inhibition studies plot of L-glutamate 
concentration versus reciprocal initial velocity at 
various fixed concentrations of L-asparagine 134 

3.34 Double-reciprocal plot of initial velocity versus L- 
aspartate concentration at fixed varied 
concentrations of L-glutamate and AMP in a constant 
ratio ( [L-glutamate] = 5 [AMP] ) 135 

3.35 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate 135 

3.3 6 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate 137 

3.37 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate Ug 

3.38 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate ^39 



3.39 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate 140 

3.40 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate 141 

3.41 Double-reciprocal plot of initial velocity versus ATP 
concentration at fixed varied concentrations of L- 
aspartate 142 

4.1 Arrhenius plot of the Vmax values for the ammonia- 
dependent reaction, varying NH3 concentration 152 

4 . 2 Arrhenius plot of the Vmax values for the ammonia- 
dependent reaction, varying ATP concentration 153 

4 . 3 Arrhenius plot of the Vmax values for the ammonia- 
dependent reaction, varying L-aspartate 
concentration 154 

4.4 Arrhenius plot of the Vmax values for the glutamine- 
dependent reaction, varying L-glutamine 
concentration I55 



XI 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 



INVESTIGATION OF THE KINETIC MECHANISM OF GLUTAMINE- AND 

AMMONIA-DEPENDENT REACTIONS OF E. coli ASPARAGINE SYNTHETASE 

B USING ISOTOPE PARTITIONING AND STEADY- STATE KINETICS 

By 

POURAN HABIBZADEGAH-TARI 
August, 1996 



Chairman: Dr. Sheldon M. Schuster 

Major Department: Biochemistry and Molecular Biology 

The kinetic mechanism of the Escherichia coli asparagine 
synthetase B was deduced from initial velocity studies. In 
addition to varying substrate concentrations in a variety of 
ratios, products and substrate analogs were used as 
inhibitors in additional studies. While these studies 
provided limitations to the possible kinetic mechanisms, the 
results were equivocal. The data were consistent with a ping 
pong mechanism with either inorganic pyrophosphate or L- 
glutamate released prior to addition of other substrates. 
Therefore, from these data, two ter-quad mechanisms, bi-uni- 
uni-ter ping-pong and uni-uni-bi-ter ping-pong, were 
possible. In order to resolve this dilema, a series of 
isotope partitioning studies of both the glutamine- and 



XII 



ammonia-dependent reactions of ASB were carried out as well. 
Major conclusions derived from the isotope partitioning 
experiments regarding the kinetic mechanism of ASB are as 
follows: 1) The enzyme catalyzes aspartate-dependent ATP 
hydrolysis, which requires no nitrogen source. 2) The binding 
and hydrolysis of L-glutamine or the binding of ammonia is 
not required prior to ATP and L-aspartate binding for the 
synthesis reaction. These results together clearly support an 
ordered bi-uni-uni-ter ping-pong mechanism for the ammonia- 
and the glutamine-dependent reactions of E. coli ASB, with 
ATP binding first and L-aspartate second. This is followed by 
release of PPi and subsequent addition of L-glutamine or 
ammonia. 

The information obtained from product inhibition studies 
was also consistent with the existence of an isomerization 
step following the release of the last product. Another very 
important observation made in this work was that the 
glutaminase reaction was shown to be occuring at the same 
time as the synthetase reaction, and in fact increasing with 
increasing concentrations of L-glutamine 

These observations resulted in a complete model for the 
glutamine-dependent AS reaction, and computer modeling was 
used to stimulate the proposed mechanism. According to the 
proposed model the reaction mechanism is quite complex, and 
is an ordered bi-uni-uni-ter ping-pong mechanism with a side 
reaction, glutaminase, for the E. coli ASB enzyme. 






XI 11 



CHAPTER 1 
INTRODUCTION 

Vauqulien and Robiquot (1806) were the first to report 
the isolation of L-asparagine, which was the first amino acid 
ever identified. The metabolic importance of this amino acid 
has emerged from numerous studies that have appeared 
describing both the anabolic and catabolic pathways from 
numerous organisms. Early on it was shown that L-asparagine 
can be hydrolyzed by L-asparaginase to yield L-aspartate and 
ammonia (Broome, 1963), and for many years this was thought 
to be the major use of L-asparagine. However, a study by 
Meister et al . (1952) had suggested that L-asparagine can 
also be degraded via asparagine transamination followed by 
amide hydrolysis. Later, Moraga et al. (1989) described the 
mode of L-asparagine catabolism via asparagine transaminase 
and co-amidase in rat liver mitochondria. In addition, 

evidence was presented that suggested the existence of an as 
yet uncharacterized pathway for asparagine catabolism in 
mitochondria. L-asparagine has also been shown to be involved 
in providing the residue necessary for linkage of 
oligosaccharides to glycoproteins (Spiro, 1969), so that 
possibly the most important fate of L-asparagine is its 
incorporation into protein (Coony and Hands chumacher, 1970) . 

The interest in asparagine metabolism was intensified 
from the finding by Kidd (1953) that a factor in guinea pig 



serum had antitumor activity. Later, Broome related the 
antitumor properties of guinea pig serum to its L- 
asparaginase activity (1963 and 1968) . Since that time, L- 
asparaginase, which catalyzes the hydrolysis of L-asparagine, 
has been used clinically to treat patients with lymphomas 
(Oettgen et al., 1970 and Ertel et al., 1979). When used on 
patients with acute lymphoblastic leukemia (ALL) , L- 
asparaginase resulted in complete remission for 40% of the 
patients (Uren et al., 1977). when L-asparaginase was used in 
combination with prednisone and vincristine, it resulted in 
95% complete remission for ALL previously untreated patients 
(Uren et al., 1977). Although L-asparaginase is a potent 
chemotherapeutic agent, several side effects have been 
reported that would limit its general utility. These side 
effects include chills, fever, nausea, life-threatening serum 
ammonia concentrations, liver dysfunction (Oettgen et al., 
1970 and Terebello et al., 1986), central nervous dysfunction 
(Land et al., 1972), and tumor resistance (Uren et al., 1977). 

The effectiveness of L-asparaginase is due to its 
ability to lower the circulating level of asparagine (Broome, 
1968). This suggests the possibility that a highly specific 
and potent inhibitor of the enzyme responsible for synthesis 
of L-asparagine, namely asparagine synthetase (AS) , might be 
effective in treating tumors. A great deal of the early work 
on AS was directed at this goal which involved screening of a 
broad range of inhibitors. Several hundreds of randomly 
selected compounds and a host of available and newly 



synthesized substrate analogs have been tested as AS 
inhibitors. Few compounds inhibited AS, and those that did, 
exhibited weak inhibition. The most promising inhibitor of 
asparagine synthetase, (3-aspartyl methylamide, increased life 

span from 31 to 7 9 percent when administered to mice bearing 
L-asparaginase resistant tumors (Uren et al., 1977). However, 
this compound was proven to be a better inhibitor of the L- 
asparaginase than of AS. No other compounds have been found 
to possess sufficient potency to warrant further study. 
Therefore, detailed structural, chemical and mechanistic 
information is essential in order to design effective 
inhibitors of AS. For example, given the success in the 
discovery of drugs based upon transition state analogs 
(Barlett and Marlowe, 1983), elucidation of the mechanistic 
details underlying the AS reaction mechanism may prove an 
alternate approach to obtaining potent AS inhibitors. 
Therefore, we have chosen to study in detail the mechanism of 
AS. 

Asparagine synthetase was demonstrated for the first 
time in Lactobacillis arabinosus (Ravel et al., 1962). The 
bacterial enzyme was shown to catalyze conversion of L- 
aspartate to (3-aspartylhydroxamate in the presence of 
hydroxylamine and ATP, and alternatively to L-asparagine in 
the presence of ammonia and ATP. 

L-Asp + ATP + NH ► L-Asn + AMP + PP m \ 

3 i v I 



Along with the adenosine triphosphate (ATP) a divalent metal 
ion, either Mn 2 + or Mg 2+ , was required. Adenosine 5'- 
phosphate and inorganic pyrophosphate (PPi) were shown to be 
products along with L-asparagine. The production of 
asparagine synthetase decreased more than 10- fold when the 
cells were grown in the presence of L-asparagine. In 
addition, the activity in vitro of the enzyme was inhibited 
by L-asparagine (Ravel et al . , 1962). 

Following the partial purification (10-fold) , the 
initial velocity and other properties were determined. The Km 
for ATP, L-aspartate and Mg 2 + were 0.2 mM, 4.2 mM and 3.5 mM, 
respectively. The pH optimum for the formation of P~ 

aspartylhydroxamate was between 6.0 and 6.5, and for the 
synthesis of L-asparagine was 8.2. The enzyme was also shown 
to catalyze an aspartate-dependent exchange of ATP and PPi 
which was inhibited by L-asparagine. L-glutamine did not 
serve as a nitrogen source. 

Asparagine synthetase from Streptococcus bovis was 
studied by Burchall et al. in 19 64. The bacterial extract was 
shown to catalyze the formation of a hydroxamate of L- 
aspartate in the presence of ATP and Mg +2 . The AS from 
Streptococcus bovis was partially purified from the extract 
(21-fold) and characterized. The bacterial enzyme was shown 
to catalyze the conversion of L-aspartate to p- 

aspartylhydroxamate in the presence of hydroxyl amine, ATP and 
Mg +2 - Ammonia could also be used to replace hydroxylamine , 
forming L-asparagine, but L-glutamine could not. ATP was 



converted to AMP and PPj_, and no ADP was detected. The Km's 

for ATP, L-aspartate, NH4 + and Mg 2+ were 4 mM, 26 mM and 4 mM 

and 45 mM, respectively. Fifty percent of the enzyme activity 
was lost when AS was incubated with iodoacetate (10 mM) , p- 

hydroxymercuribenzoate (10 mM) and silver nitrate (10 mM) . L- 
asparagine, which inhibited the enzyme activity at a 
concentration of 2 mM, was found to be a competitive 
inhibitor with respect to L-aspartate. Curiously, however, 
the enzyme synthesis was not repressed when bacterial cells 
were grown in the presence of L-asparagine. 

The presence of AS in E. coli was demonstrated by Ceder 
and Schwartz (1969a) . AS was purified 370-fold from a mutant 
of E.coli that was either deficient in L-asparaginase II or 
else its activity was inhibited by 5-diazo-4-oxo-l-norvaline 
(DONV) . The molecular weight of the enzyme was determined to 
be 80,000 by gel filtration and was shown to be stabilized by 
2-mercaptoethanol and by 10% glycerol. The asparagine 
synthesis reaction required L-aspartate, an ATP-Mg 2+ complex, 
and ammonia, with stoichiometric production of PPi and AMP. 
L-glutamine did not serve as a nitrogen source, but 
hydroxylamine could be substituted for ammonia, forming [J- 
aspartylhydroxamate instead of L-asparagine. The pH optimum 
was found to be 8.4. The enzyme, in the absence of ammonia, 
was shown to catalyze an aspartate-dependent exchange of ATP 
and PPi, which was inhibited by L-asparagine. 

Kinetic studies were performed to obtain the mechanism 
of the E. coli AS enzyme (Ceder and Schwartz, 1969b). Initial 






velocities of AS activity were determined when the 
concentration of two of the three substrates, L-aspartate, 
ATP, and ammonia were varied, keeping the third substrate 
constant. When either L-aspartate and ammonia, or ATP and 
ammonia was varied, a parallel initial velocity pattern was 
seen, characteristic of a ping-pong mechanism where a product 
(PPi) is released before ammonia binds the enzyme. The 
initial velocity pattern was intersecting when L-aspartate 
and ATP were the variable substrates, suggesting that these 
two substrates are added in a sequential manner to the 
enzyme. The enzyme was also shown to catalyze the transfer of 
18 from the fi-carbonyl group of aspartate to the phosphate 
of AMP, suggesting a (3 aspartyl-adenylate intermediate. The 
product inhibition studies indicated that the order of 
addition of ATP and L-aspartate is random, which was 
confirmed by kinetic studies of the PPJ.-ATP exchange. Initial 
rates of the 32 P-PPi exchange reaction were determined with 
varying concentrations of ATP and substrate, while 
maintaining the concentration of PPi constant. An 
intersecting pattern was observed. In both, the point of 
intersection was situated to the left of the ordinate. These 
results were consistent with the rapid equilibrium random 
mechanism, for which a general rate equation was presented. 
The mechanism of E. coli AS was reported to be bi-uni-uni-bi 
Ping-Pong, with random ATP and L-aspartate addition and 
random AMP and L-asparagine release as shown below. 






/ 



ATP Asp Asn AMP 

E-ATP £-AMP 



E-Asp 
ATP Asp 




t I 

E-ATP-Asp*-^ E-AMP-Asp4->> E-AMP-Asn 







Later, Felton et al. (1980) showed that E. coli contains two 
genes coding for asparagine synthetase, which were renamed 
asnA and asnB. Their studies showed that these genes are 
located at two points in the chromosome and that both must be 
mutated to produce an auxotroph. 

Biochemical and genetic studies were performed by 
Humbert and Simoni (1980) to determine if the E. coli genes 
were distinct or the if they were products of gene 
duplication. Two strains, asnA + asnB and asnA asnB + , were 
constructed, and the asparagine synthetic reaction of their 
extracts was characterized. Their studies showed that asnA 
gene codes for the enzyme previously characterized by Ceder 
and Schwartz (1969). The asnB gene coded for an enzyme (ASB) 
that was different from ASA. Dialyzed extracts containing ASB 
enzyme had a lower specific activity for asparagine 
synthesis. ASB was able to use L-glutamine or ammonia as the 
nitrogen source. ASB was also distinguished from ASA by its 



8 



greater lability at low temperatures and greater stability at 
high temperatures. 

The E. coli ASA, an ammonia-dependent AS, is composed of 
330 amino acids (Nakamura et al . , 1981), while the E.coli 
ASB, a glutamine-dependent AS, is composed of 554 amino acids 
(Scofield et al., 1990). No similarity can be detected 
between the amino acid sequence of E. coli ASA and E. coli 
ASB, indicating that although the two AS synthesize the same 
product, they have evolved from different sources. A highly 
conserved protein motif characteristic of Class II aminoacyl 
tRNA synthetase was found to align with a region of E. coli 
ASA. Site directed mutagenesis of some of the conserved 
residues in the motif resulted in an inactive enzyme, 
suggesting the possibility that E. coli ASA evolved from an 
ancestral aminoacyl tRNA synthetase (Hinchman et al . , 1992). 
The E. coli ASB was aligned with the protein sequence of 
human asparagine synthetase that can use both glutamine and 
ammonia as substrates. Interestingly, the two proteins were 
shown to be quite homologous (37%) with regard to their amino 
acid sequence. This indicated that the two genes may have 
evolved from a common ancestral gene (Scofield et al . 1990). 
The significant amount of similarity between the human and E. 
coli glutamine-dependent gene products might suggest that 
these highly conserved regions are critical for the enzymatic 
activity or structure in both enzymes. 

Cloning, overexpression and characterization of ASB was 
reported by Scofield et al . (1990) and Boehlein et al. 






(1994) . The recombinant ASB possessed a molecular mass of 
about 63 KDa. Addition of chloride (10 mM) to the assay 
medium increased glutamine-dependent activity ASB by a factor 
of 2 but did not affect the ammonia-dependent reaction. 
Magnesium ion (Mg 2+ ) gave the highest activity. Co 2+ , the 
only ion that could replace Mg 2+ ' supported 80 and 50% of the 
Mg 2+ -dependent ASB activity when either L-glutamine or 
ammonia was the nitrogen source, respectively. A number of 
nucleotides were also assayed as substrates for ASB. dATP and 
ATP were utilized in both reactions at a similar rate, 
whereas GTP utilization was only 15% that of ATP. The pH 
optima were determined to be 6.5-8 for the glutamine- and 
ammonia-dependent activities of ASB. 

Asparagine synthetase was also purified and 
characterized from Klebsiela aerogenes (Reitzer and 
Magasanik, 1982) and Saccharomyces cerevisiae (Ramos and 
Wiame, 1979 & 1980) . Asparagine synthetase has also been 
isolated and characterized from higher organisms such as 
chick embryo liver (Arfin, 1967), beef pancreas (Luehr and 
Schuster, 1985), and rat liver (Hongo et al . , 1978). 
Asparagine synthetase from mammalian sources was shown to 
catalyze the conversion of L-aspartate to L-asparagine in the 
presence of L-glutamine with concomitant cleavage of ATP to 
AMP and PPi as shown below. 

L-Asp + ATP + L-GIn ► |_-Asn + L-Glu + AMP + PP. (2) 



10 



Ammonia was also shown to serve as a nitrogen source in the 
absence of glutamine. When glutamine is used, a glutaminase 
activity is associated with the asparagine synthetase 
activity. This glutaminase activity was shown to occur in the 

L-GIn + H,0 ► L-Glu + NH 3 (3) 

absence and in the presence of all substrates of the 
asparagine synthetase. 

Underlying all of these previous studies of AS is the 
concept that any potent and specific inhibitor of AS could 
become a very useful chemotherapeutic agent. The failure of 
previous attempts to inhibit AS specifically is due to a 
deficiency of our understanding of the chemical and kinetic 
mechanism of the enzyme. Although very little information is 
available regarding the chemical mechanism of AS, there is a 
general agreement that aspartyl-AMP, an activated form of L- 
aspartate, is the reaction intermediate (Luehr and Schuster, 
1985, Horowitz and Meister, 1972). Nevertheless, the pathway 
by which the amide nitrogen is transferred from the L- 
glutamine to this activated complex in AS needs further 
characterization . 

The ability to use ammonia or L-glutamine as the 
nitrogen source is also found in other amidotransf erases such 
as such as glutamine phosphoribosylpyrophosphate 
amidotransf erase, GPA, (Mei and Zalkin, 1989), CTP synthetase 
(Weng et al., 1986), and GMP synthetase (Zalkin and Truit, 



11 



1977). Two types of glutamine amide transfer domains (GAT 
domains) have been identified in glutamine amidotransf erase 
enzymes. The first type shows homology to the GAT domain 
derived from the purF gene and is called a purF- type GAT 
domain. This amidotransferase subfamily includes glutamine 
phosphoribosylpyrophosphate amidotransferase, GPA, (Mei and 
Zalkin, 1982), AS (Andrulis et al., 1987 & Scofield et al., 
1990) and glucosamine-6-P synthase (Walker et al., 1984). The 
N- terminal four amino acids in these enzymes are highly 
conserved. The second type shows homology to the GAT domain 
derived from the trpG gene which includes CTP synthetase 
(Weng et al., 1986), carbamyl -phosphate synthetase (Piette et 
al., 1984) and GMP synthetase (Zalkin and Truit, 1977). A 
trpG-type GAT domain is characterized by three internal 
regions of conserved amino acids (Mei and Zalkin, 1987). 

In 1989, Mei and Zalkin reported the chemical pathway by 
which nitrogen is transferred from L-glutamine in related 
glutamine-dependent enzymes. In purF- type enzymes, most 
notably GPA (Mei and Zalkin, 1989), the N-terminal cysteine 
(Cys-1) residue appears critical for the formation of a 
covalent glutaminyl intermediate. In this family, the Cys-1 
was shown to be critical for glutamine-dependent, but not 
ammonia-dependent, activity because covalent modification of 
this residue with diazo-oxo-norleucine (DON) resulted only in 
the elimination of glutamine-dependent activity. In GPA a 
'catalytic triad' composed of Cys-1, His-101 and Asp-29 was 
proposed. Mutagenesis of these residues in GPA resulted in 



12 



the loss of glutamine-, but not ammonia-dependent activity. 
The cysteine-histidine interaction of amidotransferases 
resembles that in papain. In papain, His-159 functions as a 
general base to increase the nucleophilicity of Cys-25; an 
acidic residue is not involved in the proton shuttle (see 
Zalkin et al . , 1989). Rather, the side chain of Asn-175 is 
hydrogen bonded with imidazole N-3 of His-159 to fix its 
position, whereas imidazole N-l accepts a proton from Cys-25. 
(see Zalkin et al . , 1989). In GPA, in analogy to papain, it 
was proposed that Cys-1 participates in an amide hydrolysis 
reaction to release ammonia, and His and Asp act as general 
bases in the reaction. On the basis of the chemical 
modification and site directed mutagenesis, it was proposed 
that glutamine is converted to an acylenzyme intermediate by 
the nucleophilic attack of the Cys-1 thiolate anion on the 
primary amide. Such a reaction would release ammonia which, 
if protected from protonation, can undergo nucleophilic 
reaction. 

The human AS, also a member of the "purF" type of 
glutamine amide transfer enzyme, is characterized by the 
presence of an N-terminal cysteine followed by conserved 
glycine and isoleucine residues (See Mei and Zalkin, 1989 and 
Richards and Schuster, 1992). Site-specific mutagenesis was 
used to replace the N-terminal cysteine (Cys-1) by alanine in 
human AS. The mutation resulted in the loss of the glutamine- 
dependent AS activity, while the ammonia-dependent activity 
remained unaffected (Van Heeke and Schuster, 1989). Because 






13 



of the fact that human AS has the conserved residues defining 
the proposed catalytic triad, it was suggested (Mei and 
Zalkin, 1989) that the hydrolysis of L-glutamine to yield 
free ammonia is the basis of the glutamine-dependent activity 
in this enzyme (Fig. 1.1). Due to the lack of structural 
information concerning the location of the L-glutamine and 
ammonia binding pockets in human AS, the support for 
enzymatic generation of 'free' ammonia as a reaction 
intermediate is circumstantial. Further, the molecular 
mechanism by which ammonia is sequestered from solvent and 
retained in its unprotonated form remains an open question 
(Richards and Schuster, 1992). On the other hand, it is true 
that all asparagine synthetases, and other purF enzymes as 
well, can utilize ammonia as a nitrogen source in the absence 
of L-glutamine, showing that free ammonia not only binds 
within the enzyme active site, but also remains sufficiently 
nucleophilic to release L-asparagine from aspartyl-AMP 
(Richards and Schuster, 1992). By contrast, the E.coli ASB 
lacks the conserved histidine residue, necessary for the 
nitrogen transfer if the reaction proceeds by the accepted 
pathway in other glutamine amidotranf erases (Fig. 1.2), but 
still retains the ability to synthesize L-asparagine and 
exhibits similar specificity to human AS (Richards and 
Schuster, 1992). Based on these findings it was suggested 
that at least for the ASB enzyme, the proposed mechanism for 
nitrogen transfer in the glutamine-dependent synthesis of 
asparagine, may not occur (Richards and Schuster, 1992). This 



14 



led to an alternative chemical pathway for the nitrogen 
transfer reaction, proposed by Richards and Schuster (1992), 
which does not require the generation of free ammonia (Fig. 
1.3). According to this mechanism L-glutamine reacts directly 
with aspartyl-AMP to form an imide intermediate. Cys-1 is 
still essential for the glutamine-dependent activity because 
hydrolysis of the imide is required to produce L-asparagine 
and the acylenzyme derivative of L-glutamate. One of the 
important features of the intermediate is that the nitrogen 
retains amide character and therefore possesses little 
basicity. The imide can interact with the protein through the 
same functional groups used for interaction with L-glutamine 
and L-aspartate. In addition, the anionic form of L- 
asparagine is the leaving group which relieves the need for 
general acid catalysis by a histidine residue. Finally, such 
a reaction mechanism eliminates the possibility of diffusion 
of ammonia from the active site during the reaction and 
removes the need for additional structural features for 
maintaining ammonia in its unprotonated form (Richards and 
Schuster, 1992). The two catalytic mechanisms described here 
are fundamentally different, and efforts have been underway 
to address the differences posed by the two proposed 
mechanisms . 

Studying the kinetic mechanism, which is the focus of 
this dissertation, would allow us to draw certain conclusions 
about the order of the binding of the substrates and release 
of products. For example, if L-glutamate release occurs prior 



15 



to the binding of either ATP or L-aspartate, it would be very- 
difficult to justify the mechanistic hypothesis involving the 
imide intermediate (Richards and Schuster, 1992). Based on 
their proposal for the nitrogen transfer mechanism, L- 
glutamine reacts directly with aspartyl-AMP to form an 
asymmetric imide intermediate. Therefore, L-glutamate release 
can not happen prior to ATP and L-aspartate binding. The 
kinetic mechanism of AS has been proposed for several forms 
of AS: e.g., ASA from E. coli (Ceder and Schwartz, 
1969,1969), 6C3HED-RG1 tumor (Chou, 1970), mouse pancrease 
(Milman et al., 1980), beef pancrease (Markin et al., 1981), 
and rat liver (Hongo and Sato, 1985), and as discussed below, 
considerable controversy exists. 

On the basis of initial velocity and product inhibition 
studies, Chou (1970) suggested that the reaction catalyzed by 
AS from 6C3HED-RG1 tumor is ping-pong. He observed double 
reciprocal plots which showed parallel lines when either L- 
glutamine was varied versus ATP or when L-glutamine was 
varied versus L-aspartate. Double reciprocal plots of 
parallel lines were also observed when L-aspartate was varied 
versus ATP. Inhibition studies showed L-asparagine was a 
competitive inhibitor with respect to L-glutamine, and PPi 
was a competitive inhibitor with respect to ATP. Based on 
these observations, the following penta-uni-bi ping-pong 
mechanism was proposed for the 6C3HED-RG1 tumor line AS. 



16 



Gin Glu ATP ppj Asp AMP Asn 

i t i t t t t 



Using steady-state kinetic methods, Milman et al. (1980) 
proposed a uni-uni-bi-ter ping-pong Theorell-Chance mechanism 
for the glutamine-dependent reaction of mouse AS. Initial 
velocity and product inhibition studies were conducted with 
the glutamine-dependent reaction of the AS from mouse 
pancreas. Parallel double reciprocal plots were observed with 
L-glutamine versus either L-aspartate or ATP, while 
intersecting patterns were observed with L-aspartate versus 
ATP. These patterns were reported to be indicative of a 
hybrid ping-pong mechanism consisting of a glutaminase 
partial reaction and a sequential catalysis involving L- 
aspartate and ATP. Product inhibition studies involving the 
four products, L-glutamate, AMP, PPi and L-asparagine, were 
carried out to delineate further the kinetic mechanism. The 
patterns from these experiments were reported to be 
consistent with a hybrid uni-uni-bi-ter ping-pong Theroll- 
Chance mechanism where the glutaminase reaction occurs first. 
In other words, L-glutamine binds first followed by the 



17 



E-NH 



Asn 



ATP PPi 



AMP 



\M 



AMP Asn 



Gin Glu Asp 

LA I \Z t t 



release of L-glutamate. L-aspartate is the second substrate 
to bind followed by a Theorell-Chance step, (ATP on/PPi off) . 
AMP and L-asparagine are subsequently released in an ordered 
fashion. However, there was disagreement between the 
predicted patterns and the experimental results for AMP 
versus ATP and L-asparagine versus all three substrates. 
These discrepancies were rationalized to suggest the 
formation E.NH3.Asn and E.NH3.AMP abortive complexes. 
The kinetic mechanism of bovine pancreatic AS was 
deduced from initial velocity studies and product inhibition 
studies, of both the ammonia- and glutamine-dependent 
reactions (Markin et al . , 1981). For the glutamine-dependent 
reaction, parallel lines were observed in the double 
reciprocal plots of 1/V versus 1/ [L-glutamine] at varied L- 
aspartate concentrations, and in the plot of 1/v versus 
1/[ATP] at varied L-aspartate concentrations. Intersecting 
lines were found for the plot of 1/V versus 1/ [ATP] at varied 
glutamine concentrations. For further clarification of the 
order of substrate addition and product release, the product 
inhibition of the initial velocity, including a dual 



18 



inhibition study, was done for the glutamine-dependent AS. 
The results supported an ordered bi-uni-uni-ter ping-pong 
mechanism. According to the proposed mechanism, L-glutamine 
and ATP sequentially bind followed by the release of L- 
glutamate and the addition of L-aspartate and the release of 
PPi, AMP, and L-asparagine. The mechanism was found to be 
significantly different for the ammonia-dependent reaction. 
NH3 bound first followed by a random addition of ATP and L- 
aspartate. PPi, AMP, and L-asparagine are then sequentially 
released. From these studies, a comprehensive mechanism has 
been proposed through which either glutamine or NH3 can 
provide nitrogen for L-asparagine production from L-aspartate 
as shown below. 



Glu and NH 3 




E-ATP-Asp-NH „<*->► E-Asn-AMP-PP. 
o 



PPi AMP Asn 

t t t 



The kinetic mechanism of rat liver AS was studied by Hongo 
and Sato (1985). The initial velocity studies, with L- 
glutamine as a varied substrate and L-aspartate as a varied 



19 



changing fixed substrate, showed a series of parallel lines 
in the form of a double reciprocal plot. A parallel double 
reciprocal plot was also observed between L-glutamine and 
ATP. On the other hand, intersecting lines were obtained 
between L-aspartate and ATP. Product inhibition studies were 
also conducted. These studies were performed with a high 
concentration of Mg 2+ . The mechanism of the reaction was 
suggested to be uni-uni-bi-ter ping-pong Theorell-Chance. 
According to their mechanism, L-glutamine binds first 
followed by L-glutamate release, and L-aspartate and ATP bind 
in an ordered manner followed by ordered release of PPi, AMP, 
L-asparagine. 



Gin Glu Asp ATP PPi AMP Asn 

i t i \y t t 



When the Mg 2 + concentration was kept 0.5-2.0 mM over the ATP 
concentration, the binding of substrates after the release of 
L-glutamate was in rapid equilibrium with ordered Mg 2+ and 
random L-aspartate-ATP. 



E- 



20 

Asp MgATP 

1 J 

Gin Glu Mg / \ PPi Mg, AMP, Asn 

i 1 1 /yt t 



MgATP Asp 



Clearly there are dramatic differences in the mechanisms 
presented, and the sources of these differences are unclear. 
The differences regarding the order of substrate binding and 
product release could be due to the different enzyme sources 
used. Another potential cause for differing results may be 
the presence of certain contaminants. While it is accepted 
that the degree of enzymatic purity will not alter a kinetic 
mechanism, the presence of any contaminant that breaks down 
the substrates or the measured product could have 
substantially contributed to the discrepancies. This is 
especially important because the presence of a contaminating 
glutaminase or asparaginase activity could potentially alter 
the observations. Further, the differences between the 
results could be due to the single experimental approach 
utilized. All studies in the past relied solely on steady 
state kinetic methods, and no other technique, such as 
isotope partitioning, was employed. 



21 



The kinetic mechanism of E. coli ASB, for both the L- 
glutamine-dependent and ammonia-dependent reactions, using an 
overexpressed, stable and pure enzyme has been investigated. 
Although the techniques employed are basically those used in 
the past, the enzyme is far more pure, and highly stable. 
These studies provide us with information regarding the 
kinetic mechanism and relevant rate constants. In addition, 
the ability to use techniques such as isotope partitioning 
(trapping) , pioneered by Meister and Rose and their co- 
workers (Krishnaswamy et al . , 1962; Rose et al., 1974), 
allows us to address problems such as substrate inhibition 
and/or abortive complex formation observed with steady state 
analyses, and will provide us with information about 
catalytic competency of the enzyme-substrate complexes. The 
data presented will show that no decision could be made on 
any one mechanism proposed for glutamine-dependent reaction 
of ASB, if we were to rely only on steady state kinetics. But 
together with the help of isotope partitioning experiments, 
support is provided for an ordered bi-uni-uni-ter ping-pong 
mechanism for the glutamine-dependent reaction of E. coli 
ASB. ATP binds first and L-aspartate second. This is followed 
by release of PPi and subsequent addition of L-glutamine. The 
L-glutamate release is not required prior to the binding of 
ATP and L-aspartate. However, further studies suggest that 
the simple ordered bi-uni-uni-ter ping-pong mechanism is not 
applicable to E. coli ASB enzyme. From these data a 
comprehensive mechanism was proposed for the glutamine- 



22 



dependent AS reaction of ASB, which was examined through 
computer modeling. 









NH, H 

■HP /V^ 1 ** 

cy»i o- 



* 



AsPm 



23 




Hi»iM 







As pa 



(b) 



^oWf _^_ A* , 



H,N*^ COj 



HO OH 



H^rr^XOj' 



AMP 




N^ CO," S' H N* 

H" transfer J h 



H 
Asp» 



w 



A 



Asp» 



Fig. 1.1 (a) Currently accepted mechanism for the hydrolysis 
of L-glutamine to yield ammonia and an acylenzyme 1 by 
analogy with purF enzyme, GPA. (b) Synthesis of L-asparagine 
by reaction of ammonia with activated aspartyl derivative 2. 
(c) Hydrolysis reaction to yield L-glutamate from the 
acylenzyme 1. Residue numbering to that of human AS. This 
diagram was taken from Richards and Schuster, 1992) . 



24 



AaiB 
HAsn 

AsnB 
HAsi 

AsiB 
HAsn 



AsnB 
HAsn 



AsnB 
HAsn 



AsnB 
HAsn 



AsnB 
HAsn 



M C 



m 

liar 6 vino i k t [d] 

:[i] w a l[f] g s - d[d] 



30 



M R 



H R G P D 
H R G P D 



W S G I Y A 
A F R F Z N 



50 

S I IV D 



V N a(g] A 
P L F[g)m 



60 
Q Pi L Y 

Q ?| I R 



I Y N H 
I YNH 



QALRAEY33 
KKMQ3HFEF 



AVE 

C L S 

40 
S D - 

V N G 

N Q 3 

V K K 

90 

R Y Q 



LRKKALELSRL 
VQCLSAMKIA- 



- - [n] A I I A H 

t y(nJc C F G F 



R L 

R L 



L t 
L | 



Q t 

C K 



K F1 ? F1 






~ _ j 

M L D 



130 



140 



160 



170 



purr 

|v|cls_ 



FA: 

F A ■ 



KTHVLAV-NGE 

IIIIHC yInge 



I L 

: l 



s z 

T A 



K D A Y L I N R sl H L [g] I I [pT 
N K K V F L IG R j| T y|_g|v R [p 1 



HIK AIL VI ? V t R T : K 

aI_kJ a Ilvj t l k h s a t 



Y M G Y o[~e] H [g| [Zl Y 

F X A M t|_fJ D [gJ F (l) A 

180 

?A3S-Y|T]v) 

LxvitruJi 



w s 

P G 



Fig. 1.2 Sequence alignment of the N- terminal domains of E. 
coli ASB and human AS as deduced from oligonucleotide 
sequencing. Identical residues are boxed. The conserved 
residues in members of the purF family, (Cys-1, Asp-29 and 
His- 102) in human AS are underlined. In the mature form of 
both human AS and ASB, the N- terminal methionine is absent 
and this is therefore numbered as zero. Amino acids are 
denoted using the standards one-letter code. This was taken 
from Richards and Schuster, 1992. 






25 








JJ - Ad COj- 

• "»' HO OH -H- ^ * , 

CO," H '0 




•Asn 



CO,' 



H* transfer 

•H* 



h,0 



u 



ASDm 




CO,' M 



? o- 



Cysi o 



<s 




** A** 



o- 



Sk 



Cys, HjN-^^cOj- 

co 2 - , 

"•o 



><s 



Aspw 



Aspjj 



Fig. 1.3 Proposed mechanism for the synthesis of L- 
asparagine by E. coli ASB, via an imide intermediate 3. 
attack of the primary amide occurs directly upon activated L- 
aspartate 2. Cys-1 and asp-33 are then involved in the 
hydrolysis of the imide to yield L-glutamate and L-asparagine 
in subsequent steps, residues numbering corresponds to that 
of the Asn B gene product. This was taken from Richards and 
Schuster, 1992. 



CHAPTER 2 

INVESTIGATION OF THE MECHANISM OF E. COLI ASPARAGINE 

SYNTHETASE USING ISOTOPE PARTITIONING 



Introduction 

Asparagine synthetase B from E. coli catalyzes the 
following reactions. 

L-Asp + ATP + NH ► L-Asn + AMP + PP ( 1 ) 

3 l 

L-Asp + ATP + L-GIn ► L-Asn + L-Glu + AMP + PP ; (2) 

L-GIn + 1-1,0 ► L-Glu + NH 3 (3) 

The E. coli ASA which catalyzes strictly the ammonia- 
dependent synthesis of L-asparagine is the most extensively 
characterized in terms of kinetic and chemical mechanism. The 
order of substrate addition and product release has been 
determined, and evidence for the existence of an aspartyl-AMP 
intermediate has also been provided (Ceder and Schwartz, 
1969a & 1969b) . 

Studies on the mammalian AS also indicated that the 
enzyme produces an aspartyl-AMP intermediate (Luehr and 
Schuster, 1985, Horowitz and Meister, 1972) . Using steady 
state kinetics, the kinetic mechanism of the glutamine- 
dependent synthesis of AS was deduced for enzymes from 
different mammalian tissues (Chou, 1970, Milman et al . , 1980, 



26 



27 



Markin et al . , 1981 and Hongo and Sato, 1985). However, there 
were disagreements in the conclusions presented as discussed 
in chapter 1. The differences regarding the order of 
substrate binding and product release could be due to 
different enzyme sources used. In addition, none of the 
studies used a pure enzyme preparation. Although it is 
generally accepted that the degree of enzymatic purity will 
not alter a kinetic mechanism, the presence of any 
contaminants that break down the substrates or the measured 
product could have contributed to the variety of results 
obtained. This is especially a potential problem with 
asparaginase and glutaminase activities. The differences 
between the studies could also be due to a limited 
experimental approach, relying solely on steady state kinetic 
methods. No other technique was used. For example, the 
isotope trapping method, isotope exchange and pre-steady- 
state kinetic methods were not used. 

The kinetic mechanism of E. coli ASB, a member of the 
purF family of glutamine-dependent amidotransf erase, has not 
been studied. Cloning, overexpression and characterization of 
ASB was performed by Scofield et al . (1990) and Boehlein et 
al. (1994) . The overexpression of the enzyme allows us to 
obtain a large quantity of highly pure and stable enzyme 
which in turn offers a unique opportunity to determine 
accurately the reaction mechanism of ASB. 

This chapter describes isotope partitioning experiments 
that were performed to obtain information on the mechanism of 



28 



ASB. The isotope trapping methodology has been used in 
studies of glutamine synthetase (Meister et al., 1962), 
argininosuccinate synthetase (Rochovansky and Ratner, 1967) 
phosphofructokinase (Uyeda, 1970), hexokinase (Rose et al., 
1974, 1979,1981) and CTP synthetase (Lewis and Villafranca, 
1989). Meister and his co-workers used the technique to show 
if an activated form of L-glutamate (glutamyl-P) was an 
intermediate in glutamine synthetase. Other investigators 
used the technique to determine the order of addition of 
substrates (Rochovansky and Ratner, 1967, Uyeda, 1970 and 
Lewis, Rose et al., 1974, 1979, 1981 and Villafranca, 1989). 
In this technique, enzyme and one radioactive substrate are 
incubated (pulse) , followed by the simultaneous addition of a 
solution (chase) containing any other substrates necessary 
for reaction, as well as a large excess of unlabelled 
substrate to dilute the radioactive substrate with the 
carrier substrate, therefore diminishing the effect of 
additional catalytic turnovers. The reaction is also 
terminated as soon as possible after mixing to avoid 
utilization of free labelled substrate. The formation of a 
labelled product establishes the catalytic competency of the 
initial enzyme-substrate complex. We applied the isotope 
partitioning technique to determine the sequence of addition 
of substrates for both the glutamine- and ammonia-dependent 
reactions of AS. The technique was also employed to obtain 
the Kd for L-aspartate and ATP. 



29 



Materials and Methods 

Chemicals and Reagents 

L- [l^C(U) ] Aspartate (224.8 mCi/mmol) was purchased from 
NEN Research Products (Boston, MA). [2,8- 3 H] Adenosine 5'- 
Triphosphate (30.0 Ci/mmol) was bought from Amersham Life 
Science (England) . Scintillation Liquid ScintiVers™ II and 
trichloroacetic acid (TCA) were purchased from Fisher 
Scientific (Orlando, FL) . The ion exchange mono Q columns 
were obtained from Bio-Rad. The pyrophosphate reagent, 
containing fructose-6-phosphate kinase pyrophosphate 
dependent (PPj_-pfk) , aldolase, triosephosphate isomerase 
(TPI), and glycerophosphate dehydrogenase (GDH) , for 
following PPi production, MgCl2, ATP, L-aspartate, L- 
asparagine, L-glutamine, AMP, ammonium acetate, ninhydrin, 
ethylenediaminetetraacetic acid (EDTA) , Tris (hydroxymethyl) 
aminomethane (Tris-HCl) , Bis (2-hydroxylethyl) iminotris 
(hydroxymethyl) methane (Bis Tris) , isopropyl-1-thio-p-D- 
galactopyranoside (IPTG) , and glycerol were all purchased 
from Sigma. DE-81 anion-exchange chromatography paper was 
supplied by Whatman (Hillsboro, OR) . Dithiothreitol (DTT) was 
obtained from Promega Corporation (Madison, Wisconsin) . 













30 




Exoression 


of 


the 


Protein 


and 


Puri 


f ication 

















An overexpression vector for E. coli ASB, pET-B, has 
been constructed by Hinchman and Schuster (1994) . The E. coli 
B strain, BL2lDE3plys S (F~, ompT, rb~, mb~) was transformed 
with pET-B plasmid. Protein expression, cell culture and 
enzyme purification was carried out as described before 
(Boehlein et al., 1994). Transformed cells were plated onto 
Luria Broth agar plates supplemented with bacto-tryptone (10 
g/Liter) , bacto yeast extract (5 g/Liter) , NaCl (5 g/Liter) , 
pH 7.0, ampicillin (100 (ig/ml) , and chloroamphenicol (30 
|lg/ml) . Plates were incubated at 37°C overnight. Single 
colonies were used to inoculate fresh minimal media 
supplemented with tryptone (10 g/ liter), ampicillin (100 |ig/ 
ml), chloroamphenical (3 fig/ml), and D-glucose (0.75%). The 
cultures were grown in an environmental shaker at 37°C. When 
they reached an absorbance (?i=600 nm) of 0.7-1.0, the 

cultures were induced by adding IPTG to a final concentration 
of 1 mM. Cells were harvested after 2.5-hours by 
centrifugation at 15,000 rpm for 5 min in a Beckman Model J2- 
21 centrifuge. The supernatant fluid was discarded and the 
pellets were stored at -70°C until needed. Cells were lysed 
by vortexing the pellets in enzyme buffer (50 mM Bis-Tris pH 
6.5, 1 mM DTT, 0.5 mM EDTA, and 10% glycerol) using one-tenth 
of the original cell culture volume. DNase was added and 
cells were left on ice for 0.5 hr. The cell debris was 



31 



removed by centrifugation at 15,000 rpm for 20 min in Beckman 
centrifuge. The soluble cell extract containing L-asparagine 
synthetase B was purified by ion-exchange chromatography, as 
described previously (Boehlein et al., 1994). Glycerol was 
then added to a final concentration of 10%. The activity of 
L-asparagine synthetase B was monitored 

spectrophotometrically at 340 nm according to the following 
coupled reactions, developed by O'Brian (1976). Two moles of 
NADH are oxidized to NAD per mole of pyrophosphate produced. 
PPi + F-6-P PFi ' PFK » F-1,6-DP + Pi 
F-1,6-DP AMfiiW » GAP + DHAP 
GAP ™ » DHAP 
2DHAP + 2 3-NADH + 2 H + ^k2-glycerol-3-phosphate + 2 (3-NAD+ 
The standard conditions for assay were 50 mM Tris-HCl (pH 
8.0), 20 mM L-glutamine, 10 mM aspartic acid, 10 mM ATP, and 
17 mM MgCl 2 . The soluble cell extract was stored at -70°C 
until needed. 

Protein Concentr ation r)Pt-erm.inaH nn 

Protein concentration was measured using Bio-Rad Protein 
Assay (Bradford, 1976). Mouse immunoglobulin G was used to 
obtain a standard curve. 

Isotope Partitioning BaBfiEJiDS n fcfi with BadAoaCtJJffl Lz 

Aspartate, glut-ami np- and Ammnnia-nPnPnHp n t R P a^inn. 

A 105-ul solution of 50 mM Tris-HCl (pH 8.0), containing 
0.50 mM L -[14 C (u)] aspartate (800 cpm/nmole) , 2 mM MgCl 2 , and 



32 



1 nmole of ASB was incubated at 37°C for three minutes after 
which 62 \il chase solution was added such that the final 

concentrations of substrates were: 50 mM Tris-HCl, pH 8.0, 
5.0 mM ATP, 8.0 mM MgCl 2 , 10 mM L-glutamine, and 30 mM 

unlabelled L-aspartate. The mixture was rapidly mixed, using 
vortex, for 3 sec. after which it was quenched by addition of 
20 p.1 of 4 M TCA. A control was done in a similar manner 
except that TCA was added prior to incubation to account for 
the background from labelled L-aspartate. A blank was also 
done in which labelled L-aspartate was added to the chase 
solution. 

In experiments that included both ATP and labelled L- 
aspartate in the pulse solution, the concentration of ATP and 
MgCl2 in the pulse were 2 . mM and 3.0 mM, respectively. 

In experiments that included both L-glutamine and 
labelled L-aspartate in the pulse solution, the concentration 
of L-glutamine in the pulse was 3.0 mM. 

In experiments that included both NH3 and labelled L- 
aspartate in the pulse solution, the concentration of NH3 in 
the pulse was 100 mM. The final concentration of NH3 
following the addition of the chase solution was 165 mM. 
After quenching, the reactions were neutralized by 
addition of 10 Hi of 3 M Tris buffer (pH not adjusted) and 
centrifuged for five minutes to remove precipitated proteins. 
It was determined that this brought pH to 6.5. Aliquots (5 
HD of the reaction mixtures were spotted on DE-81 anion 
exchange chromatography paper (28 cm wide and 21 cm long) . a 



33 



mixture of unlabelled L-aspartate and L-asparagine was also 
loaded on both edges of the paper to serve as standards. When 
the spots are air dried, the paper were placed vertically in 
a chromatography tank containing distilled water as eluant. 
The chromatography was stopped after 8 hours, and the paper 
air dried. The edges, where standards were spotted, were 
excised and sites of L-aspartate and L-asparagine were 
determined by spraying with a solution of 0.5% ninhydrin in 
absolute ethanol. The bands of radioactive L-aspartate and L- 
asparagine, located according to the unlabelled standards, 
were excised and put separately into vials with ScintiVers™ 
II fluid. The radioactivity and quantity of L-aspartate and 
L-asparagine were determined by Beckman 60001C scintillation 
counter. The total radioactivity incorporated into L- 
asparagine were normalized to the same amount of enzyme used. 
The specific activity of the labelled L-aspartate (800 
cpm/nmole) was determined after taking into account dilution 
factor and the percent of quenching associated with 14 C 
(25%) , using paper chromatography. 

All experiments were done in triplicate, and the data 
collected were evaluated by taking average of the samples 
from which the background (blank, labelled L-aspartate added 
to the chase) was subtracted. 



34 



Determination of Kd for L-Asnart.Rtp 



In experiments to determine the Kd for L-aspartate, the 
following protocol was used. A 160 |il solution of 50 mM 
Tris-HCl, (pH 8.0), containing 10.0 mM ATP, 12.0 mM MgCl2, 2 
nmole ASB, and labelled L-aspartate (0.15-0.53 mM) was 
incubated at 37°C for two minutes. A 65 ]il chase solution was 
added such that the final concentrations of substrates were: 
50 mM Tris-HCl, pH 8.0, 12 . mM ATP, 15.0 mM MgCl 2 , 10 mM L- 
glutamine, and 30 mM unlabelled L-aspartate. The 
radioactivity and quantity of L-aspartate and L-asparagine 
were determined as before. 

Isotope Partitioning Exp eriments with RaHloactivp atp , 
Qlutamine- and Ammoni a- nenendpnf R^nfinns 

A 120-nl solution of 50 mM Tris-HCl (pH 8.0), containing 
0.5 mM [2,8-3 H ]ATP (1300 cpm/nmole) , 2 mM MgCl 2 , and 1 nmole 
of ASB was incubated at 37Q C for three minutes after which 65 
Hi chase solution was added such that the final 
concentrations of substrates were: 50 mM Tris-HCl, pH 8.0, 3 
mM ATP ,31 mM MgCl 2 , 10 mM L-glutamine, and 10 mM unlabelled 
L-aspartate was added. The mixture was rapidly mixed by 
vortex for 3 sec. after which it was quenched by addition of 
20 HI of 4 M TCA. A control was done in which L-aspartate was 
omitted from the chase solution to account for other 
contaminating ATPase activities. 



35 



In experiments that included both L-aspartate and 
labelled ATP in the pulse solution, the concentration of L- 
aspartate in the pulse was 2.0 mM. 

In experiments that included both L-glutamine and 
labelled ATP in the pulse solution, the concentration of L- 
glutamine in the pulse was 3.0 mM. 

In experiments that included both NH3 and labelled ATP 
in the pulse solution, the concentration of NH3 in the pulse 
was 100 mM. The final concentration of NH3 following the 
addition of the chase solution was 165 mM. 

Following the quenching, the reactions were centrifuged 
for 5 min. Aliquots (5 Hi) of the reaction mixtures were 

spotted on DE-81 anion-exchange chromatography paper. A 
mixture of unlabelled ATP, ADP and AMP was also loaded on 
both edges of the paper to serve as standards. After drying, 
the paper was developed using 1/50 saturated ammonium acetate 
(pH 2.8) as eluant. The chromatography was stopped after 3 
hours, and the paper air dried, and unlabelled nucleotides 
were visualized on the paper by UV absorbance at 254 nm. The 
bands of radioactive ADP, ATP and AMP, located according to 
the unlabelled standards, were cut, put into vials with 
ScintiVers™ II fluid. The radioactivity and quantity of AMP, 
ADP and ATP were determined by Beckman 60001C scintillation 
counter. The total radioactivity associated with AMP were 
normalized to the same amount of enzyme used. The specific 
activity of the labelled ATP (1300 cpm/nmole) was determined 
after taking into account the dilution factor and the percent 



36 



of quenching associated with tritium (90%), using paper 
chromatography . 

All studies were done in triplicate, and the data 
collected were evaluated by taking average of the samples 
from which the background was subtracted. 

Asoartate-Denendent ATP Hydrolysis 

The assay mixture (in all experiments, the reaction 
volume was 100 Ul) was consisted of the following: 50 mM 
Tris-HCl (pH 8.0), 2 mM L-aspartate, 2 mM MgCl2 , 0.5 mM [2,8- 
3 H]ATP (600 cpm/ nmole) and 0.5 nmole ASB . Assay mixtures 
were incubated at 37°C, and reactions were terminated by 
addition of 25 ul of 4 M TCA at the indicated times. A 
control was done in a similar manner except the TCA was added 
prior to the incubation to account for the background from 
labelled ATP. a second control was done in which L-aspartate 
was omitted from the reaction mixture to account for other 
contaminating ATPase activity. The specific activity of 
labelled ATP was determined as described above. 

Two other experiments were performed in the same manner 
except inorganic pyrophosphatase and/or pyrophosphate reagent 
was added to the reaction mixtures (the reaction volume was 
100 jil). Assay mixtures were treated as described before. 
The ATP Km for this reaction was obtained under the 
following conditions. The assay mixtures (100 ul) contained 
50 mM tris-HCl (pH 8.0), 1 mM L-aspartate, 2 mM MgCl 2 and 



37 



varying concentration of ATP (0.005-0.4 mM) (30,000 
cpm/reaction) . The assay mixtures were incubated 37°C for 3 
min, and reactions were terminated by addition of 15 |il of 4 

M TCA at the indicated times. The control was done in which 
L-aspartate was removed from the reaction mixture to account 
for other contaminating ATPase activity. 

Following the quenching, the reactions were treated as 
described before, and radioactivity and quantity of AMP, ADP 
and ATP were determined by Beckman 60001C scintillation 
counter. The total counts (30,000 cpm per reaction) was 
calculated by taking into account the dilution factor and the 
percent of quenching associated with tritium (90%), using 
paper chromatography. 

Theory 

Isotope trapping was used to obtain information about the 
order of addition of substrates for both the glutamine- and 
ammonia-dependent reactions of ASB. The following scheme 
illustrates the experimental procedure used. Enzyme and 
labelled substrate (*A) are incubated (pulse). This is 
followed by rapid dilution with a chase solution, containing 
a large excess of unlabelled substrate A and of the 
complementary reactants, B and C, which are allowed to react 
and stopped by a denaturant (acid or base) (Rose, 1980). 



38 




E + A 



The labelled substrate has three fates; it can dissociate 
from the EA* complex or it can dissociate from EA*BC complex 
or it can incorporate into the product. The ability to trap 
A* as P* indicates that EA is formed in a catalytically 
competent manner and that A can bind first. If, however, 
product formation requires that B and/or C binds the enzyme 
before A, as in the case of an ordered mechanism, no labelled 
product will form. The trapping of the labelled substrate as 
product also requires that dissociation of A* from any of the 
complexes, (E. A*) , or (E.A*BC) be slow enough that a 
measurable quantity of the labelled substrate can proceed 
toward product formation. Therefore, the failure to trap A* 
as P* could be due to the following: the E.A complex may not 
be catalytically competent (A must bind after B and C) , A may 
dissociate from E.A complex, or it may dissociate from E.ABC 
complex (Rose, 1980) . 



39 



Results 



Isotope Partitioning Experiments with Radioactive L- 
Aspartate. Glutamine- and Ammonia-Dependent Reactions 



Table 2.1 summarizes the results for both the glutamine- 
and ammonia-dependent AS reactions. A solution containing 1 
nmole of AS, 0.50 mM 14 C-L-aspartate and 2 mM MgCl2 was 
incubated for two minutes. Then a chase solution, containing 
a 60-fold excess of unlabelled L-aspartate and saturating 
concentrations of ATP, L-glutamine, and MgCl2, was added. 
This was followed by quenching and product analysis. When 
radioactive L-aspartate was the only substrate in the pulse, 
very little radioactive L-aspartate (0.10 + 0.03 nmole), for 
every nmole of AS, was found trapped as L-asparagine . When 
the above experiment was modified to include ATP in the 
pulse, for every nmole of AS enzyme, about 0.90 + 0.03 nmole 
of L-aspartate was trapped as labelled L-asparagine. When L- 
glutamine was included in the pulse with radioactive L- 
aspartate, about 0.12 + 0.06 nmole of L-aspartate was trapped 
as labelled L-asparagine. 

The isotope partitioning experiment with labelled L- 
aspartate was also performed for the ammonia-dependent 
reaction of AS. In the case where NH3 (100 mM) was used in 
the pulse with radioactive L-aspartate, the chase solution 
was as described above except NH3 was substituted for L- 
glutamine to a final concentration of 165 mM. L-aspartate 



40 



(0.15 ± 0.10 nmole) was trapped as radiolabelled L- 
asparagine, for every nmole of AS. When the above experiment 
was modified to include ATP in the pulse, about 0.70 ± 0.10 
nmole of L-aspartate was trapped as labelled L-asparagine. 

Using the isotope trapping methods, the Kd for L- 
aspartate was found to be 0.065 mM (±0.02). The Kd was 
obtained from a double reciprocal plot of 1/ [nmole of trapped 
L-asparagine] versus 1/L-aspartate (Fig. 2.1). The fact that 
reactivity is measured during a single catalytic turnover, 
therefore measuring the binding of L-aspartate (labelled), 
verifies that this is the Kd. 

Isotope Partitioning E xperiments with Radioacr.ive ATP. 
Glutamine- and Ammonia-Dependent Reactions 

Table 2.2 summarizes the results for both the glutamine- 
and ammonia-dependent AS reactions. A solution containing 1 
nmole of AS, 0.50 mM labelled ATP and 2 mM MgCl2 was 
incubated for three minutes. Then a chase solution was added 
to it, containing a 60- fold excess of unlabelled ATP and 
saturating concentrations of L-aspartate, L-glutamine, and 
MgCl2, followed by quenching and product analysis. When 
radioactive ATP was the only substrate in the pulse, 0.43 + 
0.10 nmole of ATP was found trapped as AMP, for every nmole 
of AS. When the above experiment was modified to include L- 
aspartate (2 mM) in the pulse, about 2.2 + 0.25 nmole of ATP 
was trapped as AMP. when L-glutamine (3.0 mM) was included in 



41 



the pulse with radioactive ATP about 0.92 ± 0.06 nmole of ATP 
was trapped as AMP. 

In the case where NH3 (100 mM) was used in the pulse 
with radioactive ATP, the chase solution was as described 
above, except 65 mM NH3 was substituted for L-glutamine, 0.2 
±0.07 nmole of ATP was trapped as AMP, for every nmole of 
AS. The amount of ATP trapped in this experiment (0.2 nmole) 
is half of the amount trapped when labelled ATP was the only 
substrate in the pulse, for glutamine-dependent reaction 
(0.43 nmole) . 

Aspartate-Dependent ATP Hydrolysis 



In the isotope partitioning experiment where both L- 
aspartate and labelled ATP were included in the pulse, for 
every nmole of AS enzyme, 2.2 ± 0.25 nmole of labelled ATP 
was trapped as labelled AMP but theoretically no more than 1 
nmole of ATP should be trapped as AMP. This suggests that ATP 
hydrolysis may be occurring in the pulse, prior to the 
addition of chase solution that contains all the substrates. 
If this is the case, it should be possible to detect labelled 
ATP as labelled AMP in the absence of any nitrogen source. 
The following experiments were performed using labelled ATP, 
as described under Materials and Methods, and formation of 
labelled AMP was measured as a function of time in the 
absence of any nitrogen source. The rate of AMP formation was 
0.3 nmole/min, which was linear with time for 15 min (Fig. 



42 



2.1) . The rate of AMP formation was increased when 
pyrophosphate reagent or inorganic pyrophosphatase was 
present (3-4 times) . No ATP hydrolysis (AMP formation) was 
observed in the control where L-aspartate was omitted from 
the reaction mixture (data not shown) , suggesting that ATP 
hydrolysis is dependent on the presence of L-aspartate. It 
was possible that the ATP hydrolysis, in the presence of L- 
aspartate, was due to contaminating ammonia present in the 
reaction mixture, allowing the synthesis reaction (ammonia- 
dependent) to occur. To account for any possible 
contaminating NH3 that could contribute to the ATP hydrolysis 
(AMP formation), the above experiment was carried out (for 40 
minutes), using unlabelled ATP, and formation of L-asparagine 
was measured as a function of time using HPLC amino acid 
analysis. Amino acid analysis is performed on an applied 
Biosystems 130 A separation system (Schuster et al., 1993). 
No L-asparagine was detected even after 15 minutes. Very 
little L-asparagine (0.03 nmole/min) was detected after 20 
minutes of incubation, suggesting that the contaminating NH3 
is responsible for the formation of only 1/10 of the AMP. If 
ATP hydrolysis was due to the presence of contaminating 
ammonia, stoichiometry would be observed between formation of 
L-asparagine and AMP. 



43 



Table 2.1 Trapping of L-aspartate from Complexes in the 
Steady State, Using ( 14 C) L-aspartate, Ammonia- and Glutamine- 
Dependent Reactions. 



Glutamine-Deoendent 



Pulse Condition 



L-Asp v 



L-Asp* + ATP 



L-Asp* + L-Gln 



nmole of (l & C) Asn 



0.10 + 0.03 



0.90 + 0.03 



0.12 + 0.06 



Ammonia-Dependent 



Pulse Condition 



nmole of (l^C) Asn 



L-Asp* + NH3 



L-Asp* + ATP 



0.15 + 0.07 



0.70 + 0.10 



Isotope trapping experiments were performed under conditions 
described in Materials and Methods. All variations were done 
in triplicate and evaluated by taking the average from which 
the background was subtracted. 



44 



Table 2.2 Trapping of ATP from Complexes in the Steady 
State, Using ( 3 H) ATP, Ammonia- and Glutamine- Dependent 
Reactions. 



Glutamine-Dependent 



Ammonia-Dependent 



Pulse Condition 



ATP' 



ATP* + Asp 



ATP* + L-Gln 



Pulse Condition 



ATP* + 



NH 3 



nmole of (- 2 .H) AMP 



0.43 + 0.10 



2.20 + 0.25 



0.92 + 0.06 



nmole of (2-K) AMP 



0.19 + 0.07 



Isotope trapping experiments were performed under conditions 
described in Materials and Methods. All variations were done 
in triplicate and evaluated by taking the average from which 
the background was subtracted. 







2 3 4 

1/[Asp] (mM-1) 



Fig- 2.1 Determination of Kd for Aspartate. A 160 ^il 
solution of 50 mM Tris-HCl, (pH 8.0), containing 10.0 mM ATP, 
12.0 mM MgCl2, 2 nmole ASB, and labelled L-aspartate (0.15- 
0.53 mM) was incubated at 37°C for two minutes. A 65 (il chase 
solution was added such that the final concentrations of 
substrates were: 50 mM Tris-HCl, pH 8 . , 12 . mM ATP, 15.0 mM 
MgCl2, 10 mM L-glutamine, and 30 mM unlabelled L-aspartate. 
Following the quenching, the reactions were treated as 
described under Materials and Methods, and radioactivity and 
quantity of L-aspartate and L-asparagine were determined as 
described before. 



46 



5- 








4. 






^\ 


Q. 
5 3 




■ 




nmole 

• 








1. 








0. 










■ i i i i i 


■ i 


. L 1 T - ' 



1 



2 3 4 5 6 7 8 9 10 11 12 13 14 15 



Time (min) 



Fig. 2.2 Rate of AMP formation as a function of time. 
The assay mixtures (100 (J.1 ) contained 50 mM Tris-HCl (pH 
8.0), 0.5 mM ATP (600 cpm/ nmole), 2 mM L-aspartate, 2 mM 
MgCl 2 and 0.5 nmole of ASB. Assay mixtures were incubated at 
370C, and reactions were terminated by addition of TCA at the 
indicated times. Following the quenching, the reactions were 
treated as described under Materials and Methods, and 
radioactivity and quantity of AMP, ADP and ATP were 
determined as described before. 



47 



Discussion 

To determine the order of substrate binding, isotope 
trapping experiments were done using either L- 
0-^C (U) ] Aspartate or [2,8-^h] ATP in the presence or absence 
of other substrates. Our results, for the glutamine-dependent 
AS reaction, revealed little trapping (10%) of L-Asp* as L- 
Asn* when radioactive L-aspartate was used in the pulse in 
the presence or absence of L-glutamine. These data suggest 
that either L-aspartate can bind the free enzyme in a 
catalytically competent manner, or that it dissociates from 
binary (E-Asp*) or the ternary complex (E-Asp*-Gln) faster 
than it goes on to form the product. When isotope 
partitioning experiments were done with ATP in addition to 
the L-Asp* in the pulse solution, 90% of E-Asp*-ATP was 
trapped as L-Asn* . The ability to trap radioactive L- 
aspartate when ATP was included in the pulse solution 
suggests that the E-Asp*-ATP complex is formed in a 
catalytically competent manner. This strongly suggests that 
ATP binds free enzyme first followed by L-aspartate binding. 
An alternative suggestion is that in the presence of ATP, the 
rate of dissociation of L-Asp* decreased compared to the rate 
of overall product formation. What is very clear from these 
observations is that the binding and hydrolysis of L- 
glutamine is not required prior to the addition of ATP and L- 
aspartate, or no labelled L-asparagine would have been 



48 



trapped in the absence of L-glutamine. This is strikingly 
different from the mechanisms for AS suggested by Chou, 1970, 
Milman et al., 1980, Markin et al., 1981 and Hongo and Sato, 
1985, who proposed that the binding of L-glutamine had to 
occur prior to ATP and L-aspartate binding, in order for the 
overall synthesis of L-asparagine to occur. 

When isotope partitioning experiments were done with 
labelled ATP, in the absence of L-aspartate or L-glutamine, 
50% of the E-ATP* complex was trapped as AMP*. This shows 
that the E-ATP* complex is formed in a catalytically 
competent manner, further suggesting that ATP binds free 
enzyme first. Under these conditions, only half of the enzyme 
bound ATP* was converted to AMP*, it is possible that ATP* 
was bound to the enzyme and partly equilibrated with the 
subsequently added unlabelled ATP, or that it dissociated 
from binary (E-ATP*) faster than it would go on to form the 
product. It is also possible that ATP* was bound to an 
additional site (an allosteric site, for which there is no 
evidence at this time), that was inhibitory to the ATP 
binding for the synthesis, therefore causing less trapping. 

Using the isotope trapping techniques, we were unable to 
obtain the Kd for ATP. To determine the Kd for ATP, the 
concentration of the labelled ATP was varied (0.01-0.5 mM) in 
the pulse. Surprisingly, no trapping was detected below 0.5 
mM ATP. it is possible that some ATP* equilibrated with the 
subsequently added unlabelled ATP, or that it dissociated 



49 



from binary (E-ATP*) faster than it would go on to form the 
product . 

When isotope partitioning experiments were done with L- 
aspartate in addition to the ATP* in the pulse solution, E- 
ATP*-Asp was trapped as AMP*. These data suggested that the 
E-ATP*-Asp complex was formed in a catalytically competent 
manner, but the amount of product was puzzling. The fact that 
"trapped" AMP* was not stoichiometric with the amount of 
enzyme (220%) present suggested that either ATP hydrolysis 
was occurring prior to the addition of chase solution or 
product formation was occurring in the time scale of the 
pulse. We attempted to determine if the net ATP hydrolysis 
observed was an activity of the synthetase itself or that of 
some other contaminant. Our first results showed that the 
production of AMP was found to be L-aspartate dependent and 
was not stimulated by aspartate-tRNA. This suggests that the 
reaction was not a contaminant and not surely aspartyl tRNA 
synthetase. The fact that very little L-asparagine was 
detected during this reaction ruled out the possibility of E. 
coli ASA, with an apparent Km for ammonia of less than 1 \m 
(unpublished data, Boehlein) , being involved. The aspartate- 
dependent hydrolysis of ATP represented in Reaction 4, 



MgATP 2 - l-a'Pa'tate „ mp pp 



which is linear with time, is only 1/100 of the overall 
reaction rate. As shown in the Figure 2.2, a value of about 



50 



3.8 nmole was reached indicating that the L-aspartate- 
dependent hydrolysis, in the 15 minutes period, was about 8- 
fold turnover of the enzyme. The effect of the presence of 
the contaminating NH3 , determined by L-asparagine formation, 
seems to be responsible for the formation of only 1/10 of 
AMP*. This would eliminate the possibility of synthesis 
reaction by ASB being responsible for the most of the AMP* 
formation. Extrapolation of the line (Fig. 2.2) predicts an 
intersection point of approximately 0.5 nmole AMP/ 0.5 nmole 
AS, stoichiometric with the amount of enzyme, further 
supporting that the reaction is not a contaminant, and that 
the chemistry is very fast compared to the release of the 
products. However, whether L-aspartate attacks ATP, forming 
aspartyl-AMP, or L-aspartate stimulates ATP hydrolysis is not 
clear at this moment. The addition of pyrophosphate reagent 
or pyrophosphatase to the reaction mixture produced some 
increase in the rate of this reaction (3-4 fold) . The 
slowness of the partial reaction to the overall synthetase 
reaction in not a limit to its acceptance as bona fide 
partial reaction of AS, because enzyme theory recognizes the 
phenomenon of "substrate synergism" (Bridger et al., 1968). 
That is the accelerating effect on a partial reaction of the 
presence of the complementary substrate, in this case L- 
glutamine, of the enzyme. The hydrolysis seems to be 
irreversible as shown in reaction 4, since it has not been 
possible to detect 32 PPi-ATP exchange for ASB (unpublished 



51 



data) , presumably because PPi and AMP dissociate from the 
enzyme during this catalytic process. 

Given the above data it was somewhat surprising that 
when L-glutamine was included in the pulse, about 90% of E- 
ATP*-Gln was trapped as AMP*. This suggests that an E-ATP*- 
Gln complex is formed in a catalytically competent manner. 
The E-ATP* complex seems to be tightly bound in the presence 
of L-glutamine since the amount of the AMP* trapped is 
stoichiometric with the amount of enzyme present. No 
stimulation of ATP hydrolysis was observed by L-glutamine 
(data not shown) . But rather, it seems that in the presence 
of L-glutamine, ATP binding to the active site is stabilized. 

The lines of evidence presented here indicate that for 
the glutamine-dependent reaction of AS the mechanism is 
ordered, with ATP binding first to free enzyme. However, we 
can not rule out some alternative ordered mechanism with L- 
aspartate binding free enzyme, because a small amount of L- 
Asp* was trapped as L-Asn* when no ATP was included in the 
pulse. 

In the case of the ammonia-dependent AS reaction, very 
little L-Asp* (15%) was trapped as L-Asn* when NH3 was 
included in the pulse solution with radioactive L-aspartate, 
suggesting that the presence of NH3 most likely increased the 
rate of product formation relative to the dissociation rate 
of L-Asp* from free enzyme. The ability to trap radioactive 
L-aspartate (70%) when ATP was included in the pulse 
solution, shows that an E-Asp*-ATP complex is formed in a 



52 



catalytically competent manner, implying that ATP has to bind 
first. When isotope partitioning experiments were done with 
labelled ATP and NH3 in the pulse, 20% of the E-ATP*-NH3 was 
trapped as AMP*. This suggests that the NH3 binding is not 
required prior to the binding of ATP* and/or L-Asp* or the 
level of AMP* and/or L-Asn* trapped should be close to the 
amount of enzyme present. The data also suggest that the 
mechanism is ordered such that ATP binds first followed by L- 
aspartate binding. 

The amount of *ATP trapped as *AMP when NH3 was included 
in the pulse with labelled ATP (0.2 nmole) , for every nmole 
AS, is less than the amount trapped when *ATP alone was in 
the pulse for the glutamine-dependent reaction (0.43 nmole). 
It is possible that NH3 (100 mM) interfered with the ATP 
binding. It is also possible that presence of L-glutamine in 
the chase, by stabilizing the ATP binding, prevented the 
dissociation of the bound *ATP from the binary (E-ATP*) 
complex, so that it would go on to product. 

Major conclusions derived from the isotope partitioning 
experiments regarding the kinetic mechanism of ASB are as 
follows : 

1. The enzyme catalyzes aspartate-dependent ATP 
hydrolysis. This partial reaction, although very slow (1/100 
of the overall rate) , provides a direct evidence for the 
mechanism of E. coli ASB, in which the hydrolysis requires no 
nitrogen source. Argininosuccinate synthetase is another 
example where such partial reaction has been observed 



53 



(Rochovansky and Ratner, 1967). The enzyme catalyzed 
conversion of citrulline to arginine involves an ATP- 
dependent condensation between citrulline and L-aspartate. 
Both L-aspartate and citrulline could induce cleavage of ATP. 
The time course of the aspartate-dependent hydrolysis 
differed from that of citrulline-dependent . The aspartate- 
dependent hydrolysis was linear over the course of experiment 
(30 min) , while the citrulline-dependent hydrolysis reached a 
maximum in 2 minutes. The aspartate-dependent hydrolysis 
reached a value that was about 3-fold turnover of the enzyme. 
The cleavage was suggested to be irreversible, since it had 
not been possible to detect an aspartate-dependent PPi-ATP 
exchange reaction. However, the citrulline-dependent 
hydrolysis reached a value that was stoichiometric with the 
amount of enzyme present. Basically, the reaction had come to 
a halt, probably because the products remained enzyme bound. 

2. The mechanism is probably ordered, with ATP binding 
first and L-aspartate second, the preferred, but not 
mandatory order. 

3. The binding and hydrolysis of L-glutamine or the 
binding of ammonia is not required prior to ATP and L- 
aspartate binding for the synthesis reaction. 

These are significantly different from what were 
reported in the past for the AS mechanism. The differences 
seem to be due to the fact that the previous investigators 
relied solely on steady state kinetic methods, and isotope 
trapping was not used. In addition, they failed to consider 



54 



other models to explain their data, as discussed in the next 
chapter. 



CHAPTER 3 

SUBSTRATE BINDING AND PRODUCT RELEASE OF ASPARAGINE 

SYNTHETASE B STUDIED BY STEADY STATE KINETICS 



Introduction 

The proposed kinetic mechanisms for the glutamine- 
dependent reaction of AS has been reported for the enzymes 
from beef (Schuster et al., 1981) and mouse (Milman et al., 
1980) pancreases, and rat liver (Hongo and Sato, 1985) , as 
discussed in chapter 1. In all three cases, steady state 
kinetic methods were used to elucidate the kinetic mechanism. 
There are dramatic differences between the data presented, 
but all three reports agree that L-glutamine is the first 
substrate to bind. Milman et al. (1980), Markin et al. (1981) 
and Hongo and Sato, (1985) all present mechanisms that start 
with a glutaminase reaction mainly because AS exhibits a 
glutaminase activity. 

There are major differences between the data presented 
in the past and that for E. coli ASB. Using isotope 
partitioning techniques, evidence was presented regarding the 
kinetic mechanism of ASB. The results suggested that, (a) the 
kinetic mechanism for glutamine- and ammonia-dependent 
reactions is preferentially ordered, with ATP binding first 
followed by addition of L-aspartate, (b) the enzyme catalyzes 
an aspartate-dependent ATP hydrolysis that requires no 



55 



56 



nitrogen source, and (c) glutamine binding and hydrolysis is 
not required prior to the binding of the other substrates. To 
determine whether the difference in the conclusions was 
associated with using a different enzyme or with the 
techniques employed, the following initial velocity 
experiments, including studies with inhibition by products 
and substrate analogs, were performed. The experimental 
approach used here was that of Frieden (1959), which also 
allowed us to distinguish the sequential mechanism from ping- 
pong mechanisms . 

Materials and Methods 

Chemicals and Reagents 

Trichloroacetic acid (TCA) was purchased from Fisher 
Scientific (Orlando, FL) . The pyrophosphate reagent for 
following PPi production, MgCl2, ATP, L-aspartate, L- 
asparagine, L-glutamine, L-glutamate, AMP, PPi, ammonium 
acetate, L-glutamic acid y-monohydroxamate, ninhydrin, 
Tris(hydroxymethyl) aminomethane (Tris-HCl) , and Bis (2- 
hydroxylethyDiminotris (hydroxymethyl) methane (Bis Tris) , 
were all purchased from Sigma. Dithiothreitol (DTT) was 
obtained from Promega Corporation (Madison, Wisconsin) . 



57 



Expression of the Protein and Purification 

Protein expression, cell culture and enzyme purification 
was carried out as described before (chapter 2) . 

Protein Concentration Determination 

Protein concentration was measured using Bio-Rad Protein 
Assay (Bradford, 1976) . Mouse immunoglobulin G was used to 
obtain a standard curve. 
Enzyme Assays 

The velocities were measured spectrophotometrically by 
assaying for PPi production (O'Brian, 1976), as described in 
chapter 2 . The assay mixture contained the following 
components: 50 mM Tris-HCl (pH 8.0), and varying amounts of 
ATP (0.2-0.5 mM) , L-aspartate (0.2-0.7 mM) , L-glutamine (0.2- 
0.7 mM) , AMP (3-15 mM) , L-glutamate (5-50 mM) , L- 
asparagine( 0.05-0. 25 mM) , and ammonium acetate (3-50 mM) . 
MgCl2 concentration was kept constant (3 mM) , unless stated 
otherwise. The volume of the total reaction mixture was kept 
at 160 |ll. All reactions were carried out at 37°C, using 7.4 
(ig of enzyme per reaction, in all experiments, L-glutamine 
solutions were freshly prepared using recrystallized L- 
glutamine (Sheng et al . , 1993). Under all experimental 
conditions initial velocity was verified. 

All studies were done in duplicate, and the data 
collected were evaluated by taking average of the samples 



58 



from which the background (representing the ATPase activity) 
was subtracted. Velocities were reported as nmole of PPi 
produced per minute per milligram of protein. The data were 
then plotted in the form of double reciprocal plot or a Dixon 
plot (Dixon, 1953) depending upon the type of experiments and 
weighted using the computer program, Ultrafit, purchased from 
Biosof t . 

When PPi was used in product- inhibition studies, the 
velocities were measured by monitoring the conversion of L- 
aspartate to L-asparagine by HPLC amino acid analysis (Sheng 
et al., 1993). The assay mixture (in all experiments, the 
reaction volume was 200 |il) was consisted of the following: 

50 mM Tris-HCl (pH 7.0), and varying amounts of ATP, L- 
aspartate, L-glutamine, and PPi. Value of pH 7.0 was chosen 
to overcome precipitation of MgCl2 by PPi. Concentration of 
MgCl2 was kept so that it would be 1 mM above ATP and PPi. 
Assay mixtures were incubated for 15 minutes at 37°C after 
addition of enzyme (1 ^g/reaction mixture) and terminated by 
addition of 50 \il of 20% (1.22 M) trichloroacetic acid (TCA) 
containing 0.2 mM L-histidine as an internal control. The 
controls were performed in a similar pattern except that TCA 
was added prior to the incubation. An aliquot of the reaction 
mixture (10 (il) was then injected into the amino acid 
analyzer. Amino acid analysis is performed on an applied 
Biosystems 130 A separation system (Sheng et al . , 1993). 
Under all experimental conditions enzyme stability and 



59 



initial velocity were verified, doing time course 
experiments . 

All studies were done in triplicate, and the data 
collected were evaluated by taking average of the samples. 
Velocities were reported as nmole of L-asparagine produced 
per minute per milligram of protein. 

Stoichiometry of PPj_ and L-Glutamate 

In this experiment the assay mixtures (100 |il) contained 

50 mM Tris-HCl (pH 8.0), 1 mM ATP, 1 mM L-aspartate, 5 mM 
MgCl2 and varying concentration of L-glutamine (0.1-20 mM) . 

The assay mixtures and the enzyme were preincubated at 37°C 
for 3 min. The reactions were initiated by the addition of 
the enzyme (3 ^ig/reaction) and were incubated 37°C for 4 min 
before being terminated by addition of 20% TCA (15 |il) . 
Modified glutaminase assay (Bernt and Bergmeyer, 1974) was 
used to measure L-glutamate concentrations. PPi production 
was measured by modifying the continuous spectrophotometric 
assay (0' Brian, 1976) to an end point assay. In this case, 
385 |ll of the coupling buffer (50 mM imidazole, pH not 
adjusted, and 20 (-ll of pyrophosphate reagent, which was 
originally reconstituted in 1 ml of ddH20) , was added to the 
reaction mixtures, following TCA kill, and incubated at room 
temperature for 30 min. The absorbance of the resulting 
solution was measured at 340 nm, and the amount of PPi 
produced in the reaction determined from a standard curve. 



60 



The ratio of L-glutamate/PPi versus concentration of L- 
glutamine was used in plotting. 

Results 
Initial Rate Studies 



For determination of the order of the addition of the 
substrates in the E. coli ASB, initial velocity studies were 
performed and substrates varied in a systematic way. In 
particular, to determine the order of addition of substrates 
in the glutamine-dependent reaction, the concentrations of 
two of the three substrates, L-aspartate, L-glutamine and ATP 
were varied while maintaining the third substrate fixed and 
subsaturating in the absence of any products (Frieden, 1959). 
Keeping the concentration of L-glutamine fixed and 
subsaturating, the plot of 1/v versus. 1/ [L-aspartate] at 
different ATP concentrations (or vice versa (data not shown) ) 
was found to be intersecting (Fig. 3 . 1A) which was confirmed 
by slope and intercept plots (Fig. 3. IB). Parallel lines were 
obtained when 1/v versus 1/ [L-Glutamine] (constant and 
subsaturating L-aspartate) was plotted at varied ATP 
concentrations, and 1/v versus 1/ [L-aspartate] (constant and 
subsaturating ATP) was plotted at varied L-glutamine 
concentrations (Fig. 3 . 2A and Fig. 3.3A, respectively). These 
were confirmed by slope and intercept plots (Fig. 3.2B and 
Fig. 3.3B). Interestingly, this suggests that a product is 



61 



released between the addition of each pair of substrates. 
Kinetic parameters determined from these data were K Asp of 
0.05 mM, K ATP of 0.05 mM, and K G i n of 0.20 mM. 

Of all the possible mechanisms available, two mechanisms that 
have been shown from the literature, to be associated with AS 
are as follows: Scheme A, the bi-uni-uni addition of 
substrates, and Scheme B, the uni-uni-bi addition of 
substrates. 



(Scheme A) 

K1A 

K-1 



k2B 
k-2 



k3 
k-3p 



Q 



K4C 
K-4 



K5 
K-5Q 



R S 

K6 
K-6R 



K7 
K-7S 



(Scheme B) 

P 

K1A 4 k2 



K-1 



k-2p 



k3B 
k-3 



K4C 
K-4 



_K5 
K-5C 



R 

▲ 



K6 
K-6R 



K7 
K-7S 



Using the method of Fromm (1975) , the steady-state initial 
velocity equations were derived for the two mechanisms shown 
above, assuming no product present. Equations 1 and 2 were 
derived from Schemes A and B, respectively. 



62 



equation 1) 1/v = 1/V (0/ [A] + 0/ [B] + 0/ [AB] + <|>/[C] + 1) 
equation 2) 1/v = 1/V ($/ [A] + <|»/[B] + <\>/ [C] + <|»/[BC] + 1) 
Equation 1 predicts that substrate C (L-glutamine) affects 
only the intercept of the 1/v versus 1/[A] or versus 1/ [B] 
(ATP and L-aspartate or vice versa) which would result in 
parallel lines, characteristic of a ping-pong mechanism, 
which indicates that the addition of each pair of substrates 
are separated by the release of a product. However, equation 
1 indicates that substrates A and B (ATP and L-aspartate or 
vice versa) affect both the slope and intercept of 1/v versus 
1/[B] and versus 1/[A] plots, respectively, resulting in 
intersecting lines, which suggests that the addition of A and 
B is sequential. Similarly we see from equation 2 that 
substrate A (L-glutamine) affects only the intercept of the 
1/v versus 1/[B] or 1/[C] plots, predicting parallel lines. 
Equation 2 also predicts that substrates B and C (ATP and L- 
aspartate or vice versa) affect both the slope and intercept 
of the plots of 1/v versus 1/[C] and versus 1/ [B] , 
respectively, resulting in intersecting lines. In other 
words, our initial rate studies agree with the theoretical 
results obtained for both equations 1 and 2! 

The ammonia-dependent AS reaction was examined 
kinetically. It was found that the plot of 1/v versus. 
1/ [ATP] at different L-aspartate concentrations, keeping the 
concentration of ammonium acetate fixed (50 mM) , shows 
intersecting lines (Fig. 3.4A). This was verified by plotting 
the slope and intercept (Fig. 3.4B). A similar pattern was 



63 



observed when 1/v versus. 1/ [ATP] at different L-aspartate 
concentrations was plotted (data not shown) . These data 
suggest that for the ammonia-dependent reaction, the addition 
of ATP and L-aspartate is sequential. The plots of 1/v 
versus. 1/ [ATP] at varied ammonium acetate concentrations, 
keeping L-aspartate constant and subsaturating (1 mM) , (Fig. 
3.5A) and of 1/v versus. 1/ [L-aspartate] at varied ammonium 
acetate concentrations, keeping ATP constant and 
subsaturating (1 mM) , (Fig. 3 . 6A) show parallel lines. This 
was confirmed by slope and intercept plots (Fig. 3 . 5B and 
Fig. 3.6B, respectively). These data suggest that the 
addition of each pair of substrates are separated by the 
release of a product. The results for the ammonia-dependent 
AS reaction are in agreement with the theoretical predictions 
of equation 1. Equation 1 predicts that NH3 (C) affects only 
the intercept of the 1/v versus 1/ [ATP] or versus 1/ [L- 
aspartate] plot (A and B or vice versa) which would result in 
parallel lines. Equation 1 also predicts that substrates ATP 
and L-aspartate (A and B or vice versa) affect both the slope 
and intercept of 1/v versus 1/ [L-aspartate] and versus 
1/[ATP] plots, respectively, resulting in intersecting lines. 
The data indicate that the addition of ATP and L-aspartate is 
sequential, therefore suggesting that ATP and L-aspartate 
will bind first, presumably forming aspartyl-AMP. This is 
followed by release of PPi (P) and addition of the NH3 . 
However, the result for the ammonia-dependent reaction would 
not be comparable with those predicted for equation 2 (see 



64 



discussion) . Kinetic parameters determined from these data 
were K Asp of 0.05 mM, K ATP of 0.05 mM, and K NH3 of 10.0 mM. 

The initial rate study of beef pancreas AS (Markin et 
al., 1981) showed intersecting lines for the plot of 1/v 
versus. 1/ATP at varied L-glutamine concentration, suggesting 
ATP and L-glutamine bind sequentially. This was completely 
different from the observations made by Milman et al., 1980, 
Sato, 1985 and by us. The double reciprocal plot of the 
velocity dependence on ATP at various constant concentrations 
of the L-glutamine was shown to be parallel (Fig. 3.2A), 
which indicates that the addition of each pair of substrates 
is separated by the release of a product. To resolve this 
difference, the following initial rate experiment was 
performed using L-glutamine and an alternative substrate, L- 
glutamic acid y-monohydroxamate (LGH) . Control experiments 
showed that LGH was a competitive inhibitor of L-glutamine 
(Ki = 0.2 mM ) (Fig. 3.7). For the initial rate experiment, 
the concentration of ATP was varied (0.1-0.7 mM) , while 
keeping L-aspartate constant and subsaturating (1 mM) , at 
fixed concentration of L-glutamine and LGH (0.2 mM) . The 
velocities were measured spectrophotometrically as described 
before. The plot of 1/v versus. 1/ATP at fixed concentration 
of L-glutamine and LGH (Fig. 3.8) shows that the substitution 
of LGH for L-glutamine did not not have any effect on the 
slope of the line, characteristic of a ping-pong mechanism. 
This indicates that the addition of ATP and L-glutamine are 
separated by the release of a product (see discussion) . 



65 



In order to determine the effect of substrate saturation 
on the kinetic behavior, the initial rate studies were also 
performed under saturating conditions of the substrates. When 
the concentration of L-glutamine was kept constant and 
saturating (20 mM) , the plot of 1/v versus. 1/ [L-aspartate] 
at different ATP concentrations (Fig. 3.9) showed parallel 
lines, suggesting that the addition of each pair of 
substrates was separated by the release of a product (see 
discussion) . in the case where L-aspartate was kept constant 
and saturating (> 2 mM) , the plot of 1/v versus. 1/ [L- 
glutamine] at varied ATP concentration showed what appears to 
be substrate inhibition (Fig. 3.10). The pattern changed from 
parallel to intersecting. In other words, the slope of the 
double reciprocal plot began to increase (but not 
intercepts) , which represents competitive substrate 
inhibition. The same type of observation became evident for 
the plot of 1/v versus 1/ [L-glutamine] at varied L-aspartate 
concentrations when ATP was kept constant and saturating (>5 
mM) . Very high ATP concentration made L-aspartate become an 
inhibitor of synthetase reaction. 

Inhibition bv Substrate Analogs 

Substrate analogs were tested as inhibitors of ASB to 
obtain more information about the substrate binding order 
(data provided with Dr. S. Boehlein) . This alternative 
approach requires that a competitive inhibitor be available 



66 



for each substrate. Experimentally, the concentration of the 
substrate to be studied is varied at fixed varied 
concentration of the analog. The remaining substrates are 
held at fixed and subsaturating concentrations. Experimental 
details are described in the table legend. The substrate 
analogs used in this set of experiments were, AMP-PNP, B- 
methyl aspartate and L-glutamic acid y-methyl ester. Table 3.1 
shows the inhibition patterns for all the substrate analogs 
tested for the glutamine-dependent reaction of ASB. Initial 
velocity studies in the presence of AMP-PNP demonstrated 
competitive inhibition with respect to ATP, suggesting it 
competes with the ATP for the same site on the enzyme. AMP- 
PNP was found to be noncompetitive with respect to L- 
aspartate and uncompetitive with respect to L-glutamine. B- 
methyl aspartate was found to be competitive with L-aspartate 
and noncompetitive with respect to ATP and L-glutamine. L- 
glutamic acid y-methyl ester was competitive with L-glutamine, 
uncompetitive with ATP and noncompetitive with L-aspartate. 
The rate equations for the effect of analogs (competitive 
inhibitors) of each substrate were derived for the two 
mechanisms (A and B) . Although there were some disagreements, 
initial rate results (Table 3.1) in most cases agreed with 
predicted patterns obtained from rate equations for both 
mechanisms (see discussion) . 



67 



Stoichiometry o f Glutamine-dependent Reaction 

E. coli ASB can function as a glutaminase (reaction 3) 
when L-aspartate and ATP are not present, showing that L- 
glutamine can be bound by the free enzyme . In order to 
determine the relevance of the glutaminase reaction to the 
glutamine-dependent synthetase mechanism, a stoichiometry 
experiment was performed. In this experiment, the 
concentration of L-glutamine was varied while keeping ATP and 
L-aspartate constant and subsaturating (1 mM) . The 
concentrations of L-glutamate and PPi produced were measured 
simultaneously. The synthesis of L-asparagine has been shown 
to be stoichiometric with PPi formation (data not shown) 
under all circumstances. Stoichiometry should also be 
observed between formation of L-glutamate and PPi if no 
glutaminase was occurring during the synthesis of L- 
asparagine. However, as shown in Figure 11, plotting the 
ratio of L-glutamate/PPi versus L-glutamine concentration 
resulted in a hyperbolic curve, approaching a plateau with 
increasing concentrations of L-glutamine. This shows that the 
L-glutamate and PPi production is non-stoichiometric, 
indicating that glutaminase reaction is occurring at the same 
time as the synthetase reaction and at a much faster rate 
than the synthetase reaction as concentration of L-glutamine 
increases, approaching approximately 2:1. 



68 



Product Inhibition Studies 

Our initial velocity studies proved somewhat 
inconclusive information regarding the order of addition of 
the substrates. To obtain additional information about the 
order of product release, product inhibition of the initial 
velocity was studied for both the L-glutamine-dependent and 
ammonia-dependent L-asparagine synthetase reactions. The 
velocities were measured spectrophotometrically by assaying 
for PPi production (O'Brian, 1976). Figures 12 through 17 and 
Table 3.2 show the product inhibition patterns and constants 
for the ammonia-dependent AS reaction. When the concentration 
of one substrate is varied, the remaining substrates are held 
at fixed concentrations: ATP, 1 mM, L-aspartate, 1 mM, and 
ammonium acetate, 50 mM. L- asparagine was found to be 
competitive with respect to ammonia (Ki = 0.08 + 0.025 mM) , 
and noncompetitive with respect to ATP and L-aspartate (Ki = 
0.190 ± 0.002 and 0.26 + 0.002 mM, respectively). AMP was 
noncompetitive with respect to all the three substrates. The 
Ki for ATP, L-aspartate and ammonia were 3 ± 0.001, 8 + 0.001 
and 15 + 0.002 mM. The fact that L-asparagine was competitive 
with ammonia, suggesting they bind to the same enzyme form, 
allows us to place L-asparagine after ammonia (Q) followed by 
AMP (R) . 

Figures 18 through 28 and Table 3.3 show the product 
inhibition patterns and constants for the glutamine-dependent 



69 



AS reaction. When the concentration of one substrate was 
varied, the remaining substrates were held at fixed and 
subsaturating concentrations (1 mM) . The MgCl2 concentration 
was kept constant (3 mM) , unless stated otherwise. L- 
asparagine was found to be competitive with respect to L- 
glutamine (Ki = 0.015 + 0.003 mM) , suggesting it binds to the 
same enzyme form as L-glutamine. L-asparagine was 
noncompetitive with respect to ATP (Ki = 0.09 + 0.001 mM) and 
L-aspartate (Ki = 0.27 + 0.001 mM) , suggesting it binds the 
free enzyme and enzyme-substrate complex. PPi was competitive 
with respect to ATP and L-aspartate (Ki = 0.05 + 0.002 and 
0.39 + 0.01 mM, respectively). L-glutamate was a poor 
inhibitor and was shown to be noncompetitive with respect ATP 
(Ki = 47 + 0.001 mM) . L-glutamate was found to be competitive 
with respect to L-aspartate (Ki = 23 + 0.001 mM) and 
noncompetitive with respect to L-glutamine (Ki = 52 ± 0.001 
mM). AMP was also a poor inhibitor. AMP was noncompetitive 
with respect to all the three substrates. The Ki for L- 
glutamine, L-aspartate and ATP were 14 + 0.001, 9 + 0.001, 
and 5 + 0.001 mM 

For further clarification of the order of product 
release, more specifically the first product, the following 
initial velocity experiments were performed. L-aspartate and 
L-glutamine concentrations were varied against each other in 
the presence of L-glutamate (50 mM) , keeping the ATP 
concentration constant and subsaturating (1 mM) . m another 
experiment, concentrations of L-aspartate and L-glutamine 



70 



were varied in the presence of PPi (0.4 mM) , while keeping 
the ATP concentration constant and subsaturating (1 mM) . If 
L-glutamate is the first product released between L-aspartate 
and L-glutamine, then a double reciprocal plot of 1/v versus 
1/ [L-aspartate] at various L-glutamine concentrations will 
produce intersecting lines in the presence of L-glutamate. If 
however, L-glutamate is not the product released between the 
addition of L-glutamine and L-aspartate, then the double 
reciprocal plot will result in parallel lines. On the other 
hand, if ppi is is the first product released between L- 
aspartate and L-glutamine, intersecting lines will be 
observed for the plot of 1/v versus 1/ [L-aspartate] , varying 
the L-glutamine concentration. Figures 29 and 3 show the 
results of 1/v versus 1/ [L-aspartate] at various the L- 
glutamine concentrations in the presence of L-glutamate or 
PPi. The parallel lines in the presence of L-glutamate (Fig. 
3.29) suggests that L-glutamate can not be the first product 
(P) released between L-aspartate and L-glutamine. Parallel 
lines were also observed in the presence of PPf (Fig 3.30) 
which suggest that P Pi cannot be the first product off (see 
discussion) . 

To obtain more information about the order of product 
release (Q, r, s) , double-inhibition studies were performed 
for the glutamine-dependent AS reaction. This set of 
experiments examines the relation between the products, 
determining whether the two products interact with each othe; 
on the enzyme's surface. For this set of experiments, the 



ir 



71 



concentration of all the substrates was held constant (1 mM) , 
and concentrations of two of the products were varied against 
each other. If L-asparagine and AMP interact with each other 
(are next to each other in the release order) , then a Dixon 
plot (1953) of 1/v versus [L-asparagine] at various AMP 
concentrations will give intersecting lines (Segel, 1975) . 
If, however, their binding is separated by another product, 
then the Dixon plot will show parallel lines. Figures 3.31, 
3.32, and 3.33 show the dual-inhibition studies of the 1/v 
versus. [L-asparagine] and [L-glutamate] at different 
concentrations of L-asparagine, and AMP, accordingly. The 
plot of 1/v versus. [L-asparagine] at varied AMP 
concentrations (Fig. 3.31) shows intersecting lines. This 
shows that the presence of L-asparagine enhances AMP 
inhibition or vice versa, therefore, indicating that they can 
combine sequentially with the enzyme. The plots of 1/v 
versus. [L-glutamate] at different AMP and L-asparagine 
concentrations (Fig. 3.32 and Fig. 3.33) revealed parallel 
lines. The presence of L-glutamate would not enhance L- 
asparagine or AMP inhibition or vice versa, which suggests 
that the products are mutually exclusive. Based on these 
observations we are now able to place the AMP and L-glutamate 
release steps (R and S, respectively) after the release of L- 
asparagine (Q) . 

The rate equation for the proposed mechanism (Scheme A) 
was derived assuming the products P, Q, R and S were present, 
and the product inhibition patterns predicted were compared 



72 



with experimental results. There are some disagreements 
between the predicted patterns and the experimental results 
Other considerations are necessary to explain the 
experimental data (see discussion) . One of the most obvious 
possibilities is the existence of an isomerization step 
following the release of the last product shown below. 



(Scheme C) 






A 


B 






K1A 




k2B 




K-1 




k-2 



P c 

k3 
k-3p 



K4C • 
K-4 



Q R 

K5 



K-5Q 



K6 
K-6R 



K7 
K-7S 



k8 
k-8 



The iso-mechanism predicts a non-competitive inhibition 
pattern between the last product and first substrate which 
would otherwise be competitive. In other words, after the 
release of S, L-glutamate, the enzyme is in a conformation 
that is not accessible to A, as is indicated by the symbol F 
(Segel, 1975). The F form has to convert back to E by a 
reaction indicated by rate constants k8/k-8. The rate 
equation for the proposed mechanism with the iso step (Scheme 
C) was also derived assuming all products were present and 
was compared with the rate equation for the same mechanism 
with no iso step (Scheme A) . The equation for the Scheme C 
was very different and much more complex than that of the 



73 



equation for Scheme A (see discussion) . The differences that 
were useful for the comparison of the two mechanisms were as 
such: 

equation 3) 1/v = 1/v «|»[RS] / [AB] ) 
equation 4) 1/v = 1/v (<|>[RS] / [AB] + $[rs] ) 
The equation for the mechanism with no iso step (equation 3) 
predicts that the RS term affects only the slope of 1/v 
versus. 1/A (or 1/B) which would result in a competitive 
pattern (lines intersecting on 1/v axis) . Similarly the 
equation for the mechanism with iso step (equation 4) 
predicts that RS term affects both the slope and the 
intercept of the plot of 1/v versus. 1/A (or 1/B) which 
results in a noncompetitive pattern (lines intersecting to 
the left of the 1/v axis) . According to our proposed 
mechanism (Scheme A), A and B are ATP and L-aspartate or vice 
versa, and R and S are AMP and L-glutamate, respectively. 

To verify if there is an isomerization step in our 
proposed mechanism leading to unusual product inhibition 
patterns, the following initial velocity experiment was 
performed: The concentration of L-aspartate was varied, at 
fixed varied concentrations of L-glutamate and AMP in a 
constant ratio ([L-glutamate] = 5 [AMP] ) , while keeping ATP 
and L-glutamine constant and subsaturating (1mm). The plot 
of 1/v versus. 1/L-aspartate (Fig. 3.34) shows intersecting 
lines whose intersection is to the left of 1/v axis 
(noncompetitive) . This result agrees with the theoretical 



74 



prediction of equation 4, supporting a proposed mechanism 
with an iso step (Scheme C) . 

Discussion 

The initial velocity patterns of the relationship between 
pairs of substrate obtained for both the ammonia- and 
glutamine- dependent reactions were similar. From these data 
we proposed the two mechanisms (A and B) . Scheme A, the bi- 
uni-uni addition of substrates, and Scheme B, the uni-uni-bi 
addition of substrates. 



(Scheme A) 

K1A 

K-1 



k2B 
k-2 



k3 
k-3p 



Q 



K4C 
K-4 



R 



K5 
K-5Q 



K6 
K-6R 



s 

4 



K7 
K-7S 



(Scheme B) 

P 

K1A 4 k2 



K-1 



k-2p 



k3B 
k-3 



Q 

K4C 4 K5 



K-4 



R 

▲ 



K-5Q 



K6 
K-6R 



s 

A 



K7 
K-7S 



The data presented for the ammonia-dependent reaction are 
only consistent with mechanism A (bi-uni-uni-bi ping-pong) 






75 



According to mechanism A, ATP and L-aspartate bind first, 
forming aspartyl-AMP. This is followed by release of PPi (P) 
and addition of NH3 . The results for the ammonia-dependent 
reaction do not support mechanism B (uni-uni-bi-bi ping- 
pong) . According to equation 2 (derived to fit mechanism B) , 
substrate A affects only the intercept of the 1/v versus 
1/[B] or 1/[C] plots, which predicts parallel lines. This 
indicates that the addition of each pair of substrates is 
separated by the release of a product. Therefore, if 
mechanism B is the correct mechanism, following the addition 
of NH3 t a product must be released prior to the addition of 
the second substrate, which is not possible. 

When the ammonia- dependent reaction was studied for the 
beef pancreatic AS (Markin et al., 1981), the kinetic 
mechanism was proposed to be significantly different from 
that of ASB. According to their previous proposed mechanism, 
NH3 bound first followed by a random addition of ATP and L- 
aspartate. When evaluating the ammonia-dependent AS reaction, 
these workers found that the plots of 1/v versus I/NH3 at 
varied L-aspartate concentrations (at saturating ATP) and 1/v 
versus I/NH3 at varied ATP concentrations (with saturating L- 
aspartate) resulted in parallel lines. This was proposed to 
indicate either a random addition of ATP and L-aspartate or 
the release of a product between their additions. No 
information was provided regarding the pattern between ATP 
and L-aspartate (with saturating NH3 ) , and no experimental 



76 



evidence was provided to support the notion that the addition 
of ATP and L-aspartate was random. 

Although our data from initial velocity studies 
supported a bi-uni-uni-bi ping-pong mechanism (mechanism A) , 
for the ammonia-dependent reaction of ASB, we were still 
unable to determine whether the addition of ATP and L- 
aspartate is ordered or random. The data from isotope 
trapping experiments clearly indicated that the mechanism is 
ordered such that ATP binds first followed by L-aspartate 
binding (see Chapter 2) . These data together support 
mechanism A (ordered bi-uni-uni-bi ping-pong) for the 
ammonia-dependent AS reaction. The rate equation and kinetic 
parameters have been worked out (Fromm, 1975). For this 
reaction the rate equation is as follows: 

equation 5) l/ v = 1/v (1/ [ABC] - [PQRS] (01 [C] + 2 [AB] + 
03 [AC] + 04 [BC] + 05 [ABC] + 6 [P] + 7 [pq] + 8 [ A p] + 9 [ C R] + 
010 [PR] + 011 [QR] + 012 [ABP] + 013 [ABQ] + i4 [APQ] + 01 5 [BQR] 
+ 016 [PQR] + 017 [BCR] + 018 [CQR] + i9 [ABCQ] + 02O[ABPQ] + 
02l[BCQR] +022[BPQR])) 

The data for the glutamine-dependent AS reaction, 
however, are consistent with both mechanisms (A and B) . 
According to mechanism A, a bi-uni-uni-ter ping-pong 
mechanism, ATP and L-aspartate sequentially bind followed by 
PPi release. This is followed by L-glutamine which is the 
last substrate to bind. According to mechanism B, a uni-uni- 
bi-ter ping-pong mechanism, L-glutamine binds first followed 
by the release of the L-glutamate. ATP is then the second 



77 



substrate to add, followed by the addition of L-aspartate or 
vice versa. 

Kinetic studies done under saturating condition provided 
evidence of substrate inhibition (Fig. 3.10). Under these 
conditions substrate inhibition was observed with ATP and L- 
aspartate. This is an artifact that is common in ping-pong 
mechanisms and has been reported for other enzymes including 
fatty acid synthetase (Katiyar et al., 1975), 5- 
enolpyruvoylshikimate-3-phosphate synthase (EPSPS) (Gruys et 
al., 1992), UDP-N-Acetylenolpyruvylglucosamine reductase 
(Dhalla et al., 1995) and nucleoside diphosphate kinase 
(Garces and Cleland, 1969). Substrate inhibition usually is 
caused by the substrates binding to the improper forms of the 
enzyme which would result in dead-end complexes. The 
inhibition effect is competitive because the substrates 
interact with the same enzyme form (A and B with E) , which is 
characterized by an increase in the slopes and not the 
intercepts of reciprocal plot. Interestingly, when the 
concentration of L-glutamine was kept constant and high (20.0 
mM) (100 x Km), a parallel pattern was seen for the plot of 
L-aspartate versus ATP (Fig. 3.9), suggesting that the 
addition of this pair of substrates are separated by the 
release of a product. If we were to accept the parallel 
pattern for the plot of L-aspartate versus ATP, and not the 
intersecting pattern obtained under nonsaturating 
concentration of L-glutamine (1 mM) , we could argue that the 
mechanism of glutamine-dependent reaction of ASB is totally 



random with respect to all the three substrates. Therefore, 
this would rule out both proposed mechanisms A and B. 
However, according to Fromm (1975), the nonvaried substrate, 
L-glutamine in this case, must be kept above its respective 
Km, but nonsaturating. This is because if it is raised to a 
saturating concentration (100 x Km), artifactual parallel 
lines may be observed in the double-reciprocal plot. In other 
words, the magnitude of either $/AB (mechanism A) or <|)/BC 
(mechanism B) would change such that the slope term 
disappears. This in fact can explain some of the 
discrepancies between our data and the data presented by 
Markin et al . , (1981). in their study, the plot of L- 
aspartate versus ATP showed parallel lines when they kept L- 
glutamine constant and saturating (16.67 mM) . 

The double-reciprocal plot of ATP versus L-glutamine, at 
constant concentrations of L-aspartate, was parallel for 
E.coli ASB (Fig. 3.3A), rat liver AS (Hongo and Sato, 1985) 
and mouse pancreatic AS (Milman et al.,1980). Yet, the beef 
pancreatic AS displayed an intersecting pattern (Markin et 
al., 1981). This discrepancy was the motivation for using an 
alternative substrate. LGH was substituted for L-glutamine in 
the initial rate studies. We showed that the plot of 1/v 
versus. 1/ATP at varied fixed concentration of L-glutamine or 
LGH (0.2 mM), resulted in parallel lines. It appeared that 
the substitution of the LGH for L-glutamine did not alter the 
magnitude of either the <|>/AB or the <))/BC, therefore, no slope 
effect become evident. In other words, according to equation 



79 



1 (derived to fit mechanism A) , the slope term <|>/AB (A = ATP 
and B = L-aspartate or vice versa) is independent of C. 
Therefore, regardless of what C is (L-glutamine or LGH) , the 
plot of 1/v versus 1/ATP would result in parallel lines. On 
the other hand, according to equation 2 (derived to fit 
mechanism B) , the slope term 0/BC (B = ATP and C = L- 

aspartate or vice versa) is independent of A. Therefore, no 
matter what A is (L-glutamine or LGH) , parallel pattern will 
be observed for the plot of 1/v versus 1/ATP, indicating that 
the addition of ATP and L-glutamine are separated by the 
release of a product. 

Substrate analogs were used to obtain more information 
about the reaction mechanism, mainly the substrate binding 
order. Rate equations for the effect of analogs (competitive 
inhibitors) of the substrates for mechanism A were derived. 
The rate equation for the effect of AMP-PNP with respect to A 
(ATP) is described as follows: 

equation 6) 1/v = l/v max + K a /V max (A) (1 ♦ i/ Ki ) + 
Kb/V max (B) + Ki a Kb/V max (A) (B) (1 + I/Ki) + K c /V max C 
The equation shows that for the double reciprocal plot of 1/v 
versus 1/ATP at different concentrations of AMP-PNP, only the 
the slope term is altered; i.e., 
equation 7) slope = K a /V max (A) (1 + i/ Ki ) + 
Ki a Kb/V max (A) (B) (1 +I/K1) 

therefore, equation 6 predicts that AMP-pnp is a competitive 
inhibitor of ATP. 



On the other hand , when B (L-aspartate) is the varied 
substrate, the double reciprocal plot at different fixed 
concentrations of AMP-PNP will show an increase in both the 
slope and intercept terms, 

equation 8) Intercept = 1/V max (1 + K a /(A) (1 + I/Ki)} 
equation 9) Slope = 1/V max (Kb + Ki a K D / (A) (1 + I/Ki)} 
and when C (L-glutamine) is the varied substrate, only the 
intercept term increases. 

equation 10) Intercept = l/V max U + K a / (A) (1 + Kj.) + 
Ki a Kb/(A) (B) (1 + I/Ki)} 

Therefore, equation 6 predicts that AMP-PNP is a 
noncompetitive inhibitor of B (L-aspartate) and an 
uncompetitive inhibitor of C (L-glutamine). These predicted 
patterns obtained from equation 6 are therefore comparable 
with the experimental results obtained with AMP-PNP with 
respect to ATP, L-aspartate and L-glutamine, respectively 
(see Table 3.1). 

The rate equation for the effect of 0-methyl aspartate 
of L-aspartate is described by eq. (11) . 

equation 11) l/ v = l/v max + K a /V max (A) + K b /V max (B) (1 + 
I/Ki) + Ki a Kb/V max (A) (B) + K c /V max C 

As can be seen from equation 11, a competitive inhibitor with 
respect to L-aspartate will show uncompetitive inhibition 
with respect to ATP and L-glutamine. However, there is some 
disagreement with the experimental result, in that P-methyl 
aspartate was found to be noncompetitive with respect to ATP 
and L-glutamine. 



81 



When the rate equation for the effect of L-glutamic acid 
y-methyl ester of L-glutamine is derived, the following 

relationship is obtained. 

equation 12) 1/v = 1/V max + K a /V max (A) + Kb/V max (B) + 

Ki a Kb/V max (A) (B) + K c /V max C (1 + I/Ki) 

The equation predicts that a competitive inhibitor with 

respect to C (L-glutamine) should be uncompetitive with 

respect ATP and L-aspartate. However, L-glutamic acid y-methyl 

ester was found to be noncompetitive with respect to L- 
aspartate. 

The rate equations were also obtained for the effect of 
analogs of the substrates for mechanism B. The rate equation 
for the effect of L-glutamic acid y-methyl ester on L- 

glutamine is described as follows: 

equation 13) 1/v = 1/V max + K a /V max (A) (1 +I/Ki) + 

Kb/V maX (B) + K c /V max (C) + KibK c /V max (B)C) 

According to the equation, the inhibitor that is competitive 

with respect to A (L-glutamine) , will be uncompetitive with 

respect to (B) ATP and (C) L-aspartate. However, the 

ejcperimental result is somewhat different in that L-glutamic 

acid y-methyl ester was found to be noncompetitive with 

respect to L-aspartate. 

The rate equation was derived for the effect of AMP-PNP 
with respect with B (ATP) as shown below: 

equation 14) 1/v = l/Vma X + Ka/Vma X (A) + Kb/Vma X (B) (1 + 
I/Ki) + Kc/Vma x (C) + KibKc/Vma X (B) (C) (1 + I/Ki) 



82 



Equation 14 predicts that a competitive inhibitor with 
respect to ATP (namely AMP-PNP) should be noncompetitive with 
respect to L-aspartate and uncompetitive with respect to L- 
glutamine. These predictions are comparable with the 
experimental results (see Table 3.1). 

The rate equation for the effect of p-methyl aspartate 

of L-aspartate is described as follows: 

equation 15) 1/v = 1/Vmax + Ka/Vmax(A) + Kb/Vmax (B) + 
Kc/Vmax(C) (1 + I/Ki) + KibKc/Vmax(B) (C) 1/Vmax 
The equation predicts that a competitive inhibitor with 
respect to L-aspartate should be noncompetitive with respect 
to ATP and uncompetitive with respect to L-glutamine. This 
disagrees with the experimental results (see Table 3.1) in 
that it was found to be noncompetitive with respect to ATP 
and L-glutamine. For most cases the experimental data agreed 
with the theoretical results, however it was not possible to 
differentiate between bi-uni-uni ter ping-pong (Scheme A) and 
uni-uni-bi ter ping-pong (Scheme B) mechanisms using this 
approach. 

AS can function as a glutaminase in the presence or 
absence of the ATP and L-aspartate. Other glutamine 
amidotransferases such as carbamyl phophate synthetase 
(Nagano et al . , 1970), cytidine triphosphate synthetase 
(Levitzki and Koshland, 1971) and glutamine 

phosphoribosylpyrophosphate amidotransf erase (Mei and Zalkin, 
1989) have also been shown to have this activity, in most 
cases, however, the relative amount of glutaminase activity 



83 



appeared to be far less than the overall reaction rate, 
except for the AS from leukemia cells that was shown to have 
higher glutaminase activity compared to synthetase (2:1) 
activity. Our data showed that the formation of L-glutamate 
was higher than that of PPi for ASB. It also showed that 
ratio of L-glutamate to PPi increased with increasing 
concentration of L-glutamine. This suggests that glutaminase 
activity is happening at the same time as synthetase 
activity, and by increasing the concentration of L-glutamine, 
the glutaminase activity increases significantly over the 
synthetase activity. 

The rate equation for the proposed mechanism A (ordered 
bi-uni-uni-ter ping-pong) was derived assuming the products 
P, Q, R and S were present, as shown below. 

equation 16) 1/v = 1/V (1/ [ABC] - [PQRS] (<|>i [C] + <|>2 [AB] + 03 
[BC] + 04 [AC] + 05 [ABC] + 06 [P] + 07[PQJ + 08 [PS] + 0g[PQR] + 
010 [PQS] + 011 [SPR] + 012tSPQR] + 013 [SQR] + 014 [AP] + 0i 5 [CS] 
+ 016 [APQ] + 017 [APB] + 018 [ABQ] + 0ig[CRS] + 020 [BCS] + 
02l[APQR] + 022tABPQ] + 023 [ABQR] + 024[BQRS] + 025[ABCQ] + 
026tCQRS] + 027tABCR] + 028tBCRS] + 029 [ABPQR] + 03o[BPQRS] + 
03l[ABCQR] + 032[BCQRS] ) ) 

Assuming Q, R, and S = 0, equation 16 becomes 

equation 17) 1/v = 1/V (0i/ [AB] + 02/ [C] + 03/ [A] + 04/ [B] 

05 + 06 [P]/ [ABC] + 014tP]/[BC] +017[P]/[C]) 

Equation 17 predicts that P (PPi) is noncompetitive with 

respect to ATP and L-aspartate and competitive with respect 

to L-glutamine which are contrary to the experimental 



84 



results. PPi was found to be competitive with respect to ATP 
(Fig. 3.21) and L-aspartate (Fig. 3.22). No definite 
inhibition pattern was obtained versus L-glutamine, however. 
Therefore, the AS mechanism is different from mechanism (A) 
in which competitive inhibition of PPi versus L-glutamine is 
the expected pattern. 

Assuming P, R, and S = 0, 
equation 18) 1/v = 1/v ((|>i/[AB] + <t>2/[C] + 03/ [A] + 4 / [b] 
+ <t>5 + 4>18[Q]/[C]) 

Equation 18 predicts that Q (L-asparagine) is competitive 
with respect to L-glutamine and uncompetitive with respect to 
ATP and L-aspartate. L-asparagine was found to be competitive 
with respect to L-glutamine (Fig. 3.18), however, it was 
found to be noncompetitive with respect to ATP and L- 
aspartate (Fig. 3.19 & Fig. 3.20). 

Assuming P, Q, and S = 0, 
equation 19) l/ v = 1/v (0i/[AB] + 02/ [c] + 3 /[A] + <J> 4 / [B] 
+ 05 + 027 [R] ) 

Equation 19 predicts that R (AMP) is uncompetitive with 
respect to all the three substrates. These are different from 
experimental results in that noncompetitive inhibition 
patterns were observed (Fig. 3.26, Fig. 3.27. & Fig. 3.28) 

Assuming P, R, and S = 0, 
equation 20) 1/v = 1/v (01/ [AB] + 2 / [C ] + 3 / [A ] + 4 / [B ] 
+ <t>5 + 015[S]/[AB] + 020 [S]/ [A]) 

According to equation 20, L-glutamate would be competitive 
with respect to ATP, noncompetitive with respect to L- 






85 



aspartate and uncompetitive with respect to L-glutamine. 
However, L-glutamate was found to be uncompetitive, 
competitive and noncompetitive with respect to ATP, L- 
aspartate and L-glutamine, respectively (Fig. 3.23, Fig. 24, 
& Fig. 3.25) . 

The rate equation for the mechanism B (ordered uni-uni- 
bi-ter ping-pong) was also derived assuming the products P, 

Q, R and S were present. 

equation 21) 1/v - 1/V (1/ [ABC] - [PQRS] (0i[PQR] + $2[PQ] + 

<(>3 [P] + <{>4[BC] + ())5[PC] + <|>6[APQR] + 07{PQRS] + +8 [APQ] + 
^9[AP] + 010 [ABC] + (|)ll[ACP] + 012 [AQR] + 013 [QRS] + <J>14 [AQ] + 
015 [A] + 016 [AC] + 017[ABCQR] + 018[BCQRS] + 0ig[CPQRS] + 
<t>20tABQR] + 02l[ABCQ] + 022 [BQRS] + <t>2 3 [PRS] + <t>24 [ABCR] + 
<>2 5[BCRS] + <|>2 6[CPRS] + 027 [ABQ] + 028 [ p QS] + 029 [PS] + 
03O[AB] + 03i[BCS] +032[CPS])) 

The product inhibition equation (equation 21) , from which the 
terms containing two or three products have been omitted, has 
the expression: 

equation 22) 1/v = 1/V (1/[ABC] (03 [P] + 04 [BC] + 05 [PC] + 
09 [AP] + 0io [ABC] + 011 [ACP] + 014 [AQ] + 015 [A] + 016 [AC] + 
021EABCQ] + 024 [ABCR] + 027 [ABQ] + 030 [AB] 031 [BCS] ) ) 

assuming A= L-glutamine, B = ATP, C = L-aspartate, P = L- 
glutamate, Q = PPi, R = AMP and S = L-asparagine. 

The rate equation was also derived for the uni-uni-bi- 
ter Theorell-Chance mechanism assuming the products P, Q, R 
and S were present. The product inhibition equation from 



86 



which the terms containing two or three products have been 

omitted, has the expression: 

equation 23) 1/v = 1/V (1/[ABC] (<|>3 [P] + 04 [BC] + 05 [PC] + 
09 [AP] + 010 [ABC] + 0H [ACP] + 014 [AQ] + 015 [A] + 016 [AC] + 
024[ABCR] + 027 [ABQ] + 030 [AB] + 03l[BCS])) 
There is basically one difference between the predicted 
patterns obtained from equation 22 (ordered uni-uni-bi-ter 
ping-pong) and those from equation 23 (uni-uni-bi-ter 
Theorell-Chance) . According to the ordered mechanism 
(equation 22) , PPi should be noncompetitive with respect to 
ATP, whereas according to the Theorell-Chance mechanism 
(equation 23), it should be competitive with respect to ATP. 
Although PPi was shown to be competitive with respect to ATP 
(Fig. 3.21), other disagreements with Theorell-Chance 
mechanism were found to exist. 

The product inhibition studies did not allow us 
unequivocally to rule out any of the proposed mechanisms. 
Yet, they provided information as to where some of the 
products can be placed in the scheme. L-asparagine was found 
to be competitive with respect to L-glutamine (competing for 
the same enzyme form) , therefore they have to be next to each 
other. PPi was competitive versus ATP and L-aspartate, which 
can be placed following the addition of these two substrates. 
To determine the product released between the addition of L- 
aspartate and L-glutamine, initial velocity experiments were 
performed where L-aspartate and L-glutamine were varied in 
the presence of L-glutamate or PPi. The parallel lines in the 



87 



presence of L-glutamate (Fig. 3.29) suggests that L-glutamate 
cannot be the first product (P) released between L-aspartate 
and L-glutamine. However, given the fact that L-glutamate is 
a poor inhibitor (Ki » 20 mM) , this can also suggest that L- 
glutamate cannot bind tightly enough for the reverse reaction 
to occur. Parallel lines were also observed in the presence 
of PPi (Fig 3.30) which suggest that PPi cannot be the first 
product off either. However, this can also suggest that PPi 
binds the enzyme such that the reverse reaction can not take 
place. In other words, it can not form the complex that is 
catalytically competent for the reverse reaction. The double 
inhibition studies were performed to determine the 
relationship between the products, therefore trying to define 
their release order. The results in Figure. 3.31 show that 
the presence of L-asparagine enhances AMP inhibition. This 
suggests that they combine sequentially with the enzyme, 
therefore they must be next to each other (Segel, 1975) in 
order. However, the presence of L-glutamate shows no 
enhancement to either L-asparagine or AMP inhibition (Fig. 
3.32 and 3.33), suggesting that their binding is separated by 
another product or some other step. 

Our data from initial velocity studies supported a bi- 
uni-uni-bi ping-pong mechanism (mechanism A) , for ammonia- 
dependent reaction of ASB. Initial velocity, product 
inhibition (single or double) studies were performed in an 
attempt to come up with a mechanism, for glutamine-dependent 
AS reaction. Initial velocity experiments were also performed 



88 



in the presence of either L-glutamate or PPi to obtain 
information about the first product released in the mechanism 
(Fig. 3.28 and 3.29), from which similar results (parallel 
pattern) were obtained. Studies using saturating 
concentrations of the substrates, and the use of alternative 
substrate helped to explain some of the discrepancies that 
existed between the data presented in the past and this work. 
These studies, in general, showed us that the mechanism is 
ping-pong and not sequential. However, it became quite evident 
that relying solely on steady state kinetics, neither one of 
the mechanisms (A or B) could be ruled out. This is where the 
importance of isotope trapping experiments become obvious. 
The lines of evidence presented in chapter 2 indicated that 
for the glutamine-dependent reaction the mechanism is 
ordered, ATP being first and L-aspartate second. This is 
followed by release of PPi and subsequent addition of L- 
glutamine. Initial velocity studies, on the other hand, 
showed us that the mechanism is ping-pong and not sequential. 
These results together clearly support an ordered bi-uni-uni- 
ter ping-pong mechanism for the glutamine-dependent reaction 
of E. coli ASB. The steady-state initial velocity rate 
equation, containing all rate constants, for the proposed 
mechanism (A) in the absence of products is as follows: 
equation 24) l/ v = K ia K mb / (v max [AB] ) + K mc / (v max [C ] ) + 
Kma/(V max [A]) + Kmb/fVmax [b] ) + 1/ (v max ) 
Where, 



89 



K ma a k3 k5 k6 k7/(kl (k5 k6 k7 + k3 k6 k7 + k3 k5 k7 + k3 k5 

k6) 

Kmb = k5 k6 k7 (k3 + k-2)/(k2 (k5 k6 k7 + k3 k6 k7 + k3 k5 k7 

+ k3 k5 k6) 

K mc = k3 k6 k7 (k5 + k-4)/(k4 (k5 k6 k7 + k3 k6 k7 + k3 k5 k7 

+ k3 k5 k6) 

KmbKia = k5 k6 k7 k-1 (k3 + k-2)/kl k2 (k5 k6 k7 + k3 k6 k7 + 

k3 k5 k7 + k3 k5 k6) 

Vmax = k3 k5 k6 k7 / (k5 k6 k7 + k3 k6 k7 + k3 k5 k7 + k3 k5 

k6) [Etotal] 

A = ATP, B = L-aspartate, and C = L-glutamine 

We can now place L-asparagine release after the addition 
of L-glutamine, which is in turn followed by the release of 
AMP. Yet, as discussed earlier, there are disagreements 
between the predicted patterns and the experimental results. 
The predicted noncompetitive inhibition of PPi versus ATP and 
L-aspartate are contrary to the observed competitive 
inhibition (Fig 3.21 and 3.22). There were other 
disagreements between the predicted patterns and experimental 
results for AMP and L-glutamate which can be due to their 
poor ability to inhibit. Another possibility is the existence 
of an isomerization step (Scheme C) . The rate equation for 
the proposed mechanism with the iso step was also derived, 
assuming the products P, Q, r and S were present, 
equation 25) 1/v = 1/v (1/ [ABC] - [PQRS] (0i[PQ RS ] ♦ ^[PQR] 
+ 4>3 [PQ] + 04 [BC] + 05 [ P ] + 0g [C ] + (|) 7 {APQRS] + 8 [APQR] + 
<t>9fAPQ] + 0io [AP] ♦ 0ii [AC] + 01 2 [ABPQRS] + 13 [BPQRS] * 



90 



(J>14[ABPQR] + 0i5tABPQ] + fag [ABP] + ())17[ABC] + 018 [QRS] + 
0ig[ABQRS] + <|)20[BQRS] + <|>2l[ABQR] + <J>22 [ABQ] ) + 023 [AB] + 
4>24tABCQRS] + 025fBCQRS] + 026[CQRS] + 027 [ABCQ] + 028 [PRS] + 
029 [CRS] + 03O[ABCRS] + 03i[BCRS] + 032fABCR]) + 033 [PQS] + 
034 [CS] + 03 5 [ABCS] + 036 [BCS] + 037 [PS])) 

We focused on the terms that were not only different in the 
two equations, but also could be used to differentiate the 
two mechanisms, with and without the iso step (Scheme A and 
C) . The iso-mechanism predicts a non-competitive inhibition 
pattern between the last product and first substrate which 
would otherwise be competitive. The results from figure 3.34, 
which is the plot of 1/v versus 1/L-aspartate at fixed varied 
concentrations of L-glutamate and AMP in a constant ratio 
( [L-glutamate] = 5 [AMP]), are compatible with the predicted 
pattern from equation 4 (simplified version of the equation 
13), therefore suggesting that there is an iso step following 
the release of the last product (Scheme C) . 

One other important kinetic property of the E. coli ASB 
that was examined in this chapter was the glutaminase 
reaction. The reaction was shown to be occurring at the same 
time as the synthetase reaction and seemed to be increased 
with an increasing concentration of L-glutamine. 

Based on these results we propose the following 
mechanism for the glutamine-dependent AS reaction (Scheme D) : 



91 




PP Gin Asn AMP Glu 

k5^ |^ k7^A^ k9^A^k1 



Gin Glu n-i 

k15 . I.** * ■.«« .3 




According to this mechanism, the simple ordered bi-uni-uni- 
ter ping-pong mechanism is not applicable to E. coli ASB 
enzyme. We have two different reactions happening at the same 
time, glutaminase and synthetase. It has also been shown that 
ATP stimulates the glutaminase (Boehlein et al., 1994). 
Therefore, it is very likely that by increasing the 
concentration of the substrates, the enzyme preferentially 
goes through L-glutamine hydrolysis and not the asparagine 
synthesis reaction, causing what will appear kinetically as 
substrate inhibition (Cleland, 1983). The steady-state 
initial velocity rate equation, containing all rate 
constants, for the proposed mechanism in the absence of 
products is as follows: 

equation 26) v = ( (kl k3 k5 k7 k9 kll kl3) (kl6 + kl7) kl9 
[ABC] + k2 (k4 + k5) k7 k9 kll kl3 kl5 kl7 kl9 [C 2 ] + k3 k5 
k7 k9 kll kl3 kl5 kl7 kl9 [B C 2])/k2 k7 k9 kll kl3 kl9 (k4 + 
k5) (kl6 + kl7) [C] + k2 k4 k8 kll kl3 kl5 kl7 kl9 [C] + k2 
k5 (k8 + k9) kll kl3 kl5 kl7 kl9 [C] + k3 k5 k7 k9 kll kl3 kl9 



92 



(kl6 + kl7) [BC] + k3 k5 (k8 +k9) kll kl3 kl5 kl7 kl9 [BC] + 
kl k7 k9 kll kl3 kl9 (k4 + k5 ) (kl6 + kl7 ) [AC] + kl k3 k5 
(k8 + k9) kll kl3 (kl6 + kl7) [AB] + kl k3 k7 k9 kll kl3 (kl6 
+ kl7) [ABC] + kl k3 k5 k7 kll kl3 (kl6 + kl7) kl9 [ABC] + kl 
k3 k5 k7 k9 kl3 (kl6 + kl7) kl9 [ABC] + kl k3 k5 k7 k9 kll 
(kl6 + kl7) kl9 [ABC] + kl k3 k5 k7 k9 kll kl3 kl7 [ABC] + k4 
k7 k9 kll kl3 kl5 kl7 kl9 [C 2 ] + k2 k4 k7 kll kl3 kl5 kl7 kl9 
[C 2 ] + k2 (k4 + k5) (k7 k9 kl3 kl5 kl7 kl9 [C 2 ] + k2 (k4 +k5) 
k7 k9 kll kl5 kl7 kl9 [C 2 ] + k2 (k4 +k5) k7 k9 kll kl3 kl5 
kl9 [C 2 ] + k2 (k4 + k5) k7 k9 kll kl3 kl5 kl7 [C 2 ] + k3 k7 k9 
kll kl3 kl5 kl7 kl9[BC 2 ] + k2 k5 k7 kll kl3 kl5 kl7 kl9 [BC 2 ] 
+ k3 k5 k7 k9 kl3 kl5 kl7 kl9 [BC 2 ] + k3 k5 k7 k9 kll kl5 kl7 
kl9 [BC 2 ] + k3 k5 k7 k9 kll kl3 kl5 kl9 [BC 2 ] + k3 k5 k7 k9 
kll kl3 kl5 kl7 [BC 2 ] 
A = ATP, B = L-aspartate and C = L-glutamine. 

Due to the complexity of the mechanism, we were unable 
to predict any pattern or to design an experiment to test it. 
However, computer modeling with the program Quatro, was used 
to examine the proposed mechanism through simulation. 
Arbitrary numbers were assigned for the substrates (A, B and 
C) and the rate constants, and the data were plotted in the 
form of double reciprocal plot. 

Assuming all the rate constants (ki to kig) = 1, the 
plot of 1/v versus. 1/ [ATP] at different L-aspartate 
concentrations (constant L-glutamine) will result in what 
appears to be substrate inhibition, in other words, the 
lines, instead of being intersecting as was shown before 



93 



(Fig. 3.1), show slopes increasing with L-aspartate (Fig. 
3.35), and this is true whether L-glutamine is equal to 1 or 
equal to 20. 

Assuming kis = (k on for L-glutamine for the side 
reaction) , therefore eliminating any term that has this rate 
constant, equation 23 becomes equation 1 (derived to fit a 
simple mechanism, which is ordered bi-uni-uni-ter ping-pong) . 
Therefore, the plot of 1/v versus. 1/ [ATP] at different L- 
aspartate concentrations (constant L-glutamine) will result 
in intersecting lines (Fig. 3.36). This is true whether L- 
glutamine is equal to 1 or equal to 20. 

As we increase k i5 (0.001 to 0.005), Keeping L-glutamine 
constant and high (20), the plot of 1/v versus. 1/ [ATP] at 
different L-aspartate concentrations will gradually change 
from intersecting lines to parallel lines (Fig. 3.37). 
However, by increasing kl5 (> 0.005) we start seeing what 
appears to be substrate inhibition. On the other hand, if we 
keep kis constant (0.002), but increase L-glutamine (from 1 
to 20), the plot of 1/v versus. 1/[ATP] at different L- 
aspartate concentrations will gradually change from 
intersecting lines to parallel lines (Fig. 3.38 through 
3.40). This parallel pattern was observed experimentally 
(Fig. 3.9), when ATP was varied at fixed varied concentration 
of L-aspartate, keeping L-glutamine constant and saturating 
(20 irtM). According to Fromm (1975), the nonvaried substrate, 
L-glutamine in this case, must be kept above its respective 
Km, but nonsaturating. This predicts that if L-glutamine is 



94 



raised to a saturating level (100 x Km) , artif actual parallel 
lines may be observed in the double-reciprocal plot, however, 
the literature offers no explanation as to why this will 
happen. Our modeling strongly suggests one reason for the 
proposed results. By increasing L-glutamine concentration or 
in turn by increasing kl5 (k n for L-glutamine for the side 
reaction) , the enzyme preferentially goes through L-glutamine 
hydrolysis and not the asparagine synthesis reaction, causing 
apparent substrate inhibition. 

In further modeling, keeping L-glutamine constant and 
subsaturating (1 mM) , the plot of 1/v versus. 1/ [ATP] at 
different L-aspartate concentrations will change from 
intersecting lines to parallel lines only when kl5 is 0.05 
and higher (not shown). On the other hand, assuming kis = 0, 
the plot of 1/v versus. 1/[ATP] at different L-glutamine 
concentrations (constant L-aspartate) will result in 
intersecting lines (plot not shown) . As we increased ki5, 
keeping L-aspartate constant and high (5) , the plot of 1/v 
versus. 1/ [ATP] at different L-glutamine concentrations will 
still show parallel lines (as observed experimentally (Fig 
3.2A & 3.3A) ) . 

The computer modeling was also used to examine the 
simple mechanism, ordered bi-uni-uni-ter ping-pong (Scheme 
A). Keeping L-glutamine constant and subsaturating (1 mM) , 
the plot of 1/v versus. 1/ [ATP] at different L-aspartate 
concentrations will change from intersecting lines to 
parallel lines (as observed experimentally (Fig 3.2A & 3.3A)) 



95 



only when either ks or k6 or k7 is a negative number (Fig. 
3.41), a situation which is not possible experimentally. 

Using the computer modeling, we were able to examine the 
proposed mechanism (Scheme D) . We were able to show the 
change of pattern from intersecting lines to parallel lines, 
further supporting that the proposed mechanism (Scheme D) is 
correct. According to the mechanism, the simple ordered bi- 
uni-uni-ter ping-pong mechanism would not be applicable to E. 
coli ASB enzyme. This enzyme catalyzes two different 
reactions at the same time, a glutaminase and an asparagine 
synthetase. It is now clear that by increasing the L- 
glutamine concentration, therefore pushing the enzyme through 
an alternative path, glutaminase, one observes substrate 
inhibition, in which infinite substrate gives a finite, but 
reduced rate. The mechanism of asparagine synthetase, 
therefore is an ordered bi-uni-uni-ter ping-pong mechanism 
with a side reaction, glutaminase. 



96 



0.012- 






j/ 


0.010- 








0.008- 






^y'/^^^^ 


1/V 5.006- 




y£^ 


/x^^^x 


0.004- 


y*A 






0.002- 








n nnn 








u.uuu 


T~ ~T 


~r i 


I 1 1 I 1 I 



01 23456789 10 

1/[L-Asp] (mM-1) 



Fig. 3.1A Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of ATP. Each initial velocity is the average 
of two experiments. The L-aspartate concentrations were 0.1, 
0.2, 0.3, 0.4, 0.5 and 0.7 mM at various fixed concentrations 
of ATP: (plus) 0.2 mM, (cross) 0.3 mM, (square) 0.4 mM, 
(lower triangle) 0.5 mM, (open circle) 0.7 mM. The 
concentrations of L-glutamine and MgCl2 were maintained at 1 
and 3 mM, respectively. 7.4 ug of enzyme per assay was used. 



0.0020H 



Slope & 
Intercept 1.0015- 



0.0010- 



97 



□ 



□ 



+ 



□ 



1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fig. 3. IB plot of the reciprocal-fixed variable 
substrate ATP versus slope (plus) and intercept (square) from 



98 



1/v 



0.014- 



0.012- 



0.010- 



0.008- 



0.006 



0.004- 



0.002 



0.000 




3456789 10 

1/[Gln] (mM-1) 



Fig. 3.2A Double-reciprocal plot of initial velocity 
versus L-glutamine concentration at various concentrations of 
ATP. Each initial velocity is the average of two experiments. 
The L-glutamine concentrations were 0.1, 0.3, 0.5, 0.7 and 
0.9 mM at various fixed concentrations of ATP: (plus) 0.1 mM, 
(cross) 0.2 mM, (square) 0.3 mM, (lower triangle) 0.5 mM. The 
concentrations of L-aspartate and MgCl2 were 1 and 3 mM, 
respectively. 7.4 ng of enzyme per assay was used. 






99 



0.005- 



0.004- 



Slope & 0003 
Intercept 

0.002 
0.001 - 



0.000 



X 



0123456789 10 

1/[ATP] (mM-1) 



Fig 3 2B plot of the reciprocal-fixed variable 
substrate ATP versus slope (plus) and intercept (cross) from 
Fig.3.2A. 



100 



1/v 



0.008 



0.006 



0.004 



0.002- 




0.000 



0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[Asp] (mM-1) 



Fig. 3.3A Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-glutamine. Each initial velocity is the 
average of two experiments. The L-aspartate concentrations 
were 0.2, 0.3, 0.4, 0.5 and 0.7 mM at various fixed 
concentrations of L-glutamine: (plus) 0.2 mM, (cross) 0.3 mM, 
(square) 0.4 mM, (open circle) 0.5 mM, (lower triangle) 0.7 
mM. The concentrations of ATP and MgCl2 were 1 and 3 mM, 
respectively. 7.4 p.g of enzyme per assay was used. 



101 



0.004 



0.003 



Slope & 
intercept J002 _ 



0.001 - 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 



1/[Gln] (mM-1) 



Fig. 3.3B plot of the reciprocal-fixed variable 
substrate L-glutamine versus slope (plus) and intercept 
(cross) from Fig. 3.3A. 



102 



1/v 



0.020 



0.015 



0.010 



0.005- 



0.000 




3 4 5 6 7 

1/[ATP] (mM-1) 



Fig. 3.4A Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. Each initial velocity is the average of two 
experiments. The ATP concentrations were 0.1, 0.2, 0.3, 0.4, 
0.5 and 0.7 mM at various fixed concentrations of L- 
aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.4 mM, (lower triangle ) 0.5 mM. The 
concentrations of NH3 and MgCl2 were 50 and 3 mM, 
respectively. 7.4 ug of enzyme per assay was used. 






103 



0.006- 



0.005 



Slope & 0004 
Intercept 



0.003- 



0.002- 



0.001 - 




4 5 6 7 

1/[Asp] (mM-1) 



10 



Fig. 3.4B plot of the reciprocal-fixed variable 
substrate L-aspartate versus slope (plus) and intercept 
(cross) from Fig. 3.4A. 



104 



1/V 



0.010- 



0.008 



0.006- 



0.004 



0.002 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fig. 3.5A Double-reciprocal plot of initial velocity 
versus ATP _ concentration at various fixed concentrations of 
NH3 . Each initial velocity is the average of two experiments 
The ATP concentrations were 0.2, 0.3, 0.4, 0.5 and 0.7 mM at 
various fixed concentrations of NH3 : (plus) 3.0 mM, (cross) 
6.0 mM, (square) 12.0 mM, (open circle) 24.0 mM, (lower 
triangle) 3 6.0 mM. The concentrations of L-aspartate and 
MgCl2 were 1 and 3 mM, respectively. 7.4 ug of enzyme per 
assay was used. 



105 



0.008- 



0.006- 



Slope & 
Intercept 



0.004- 



0.002 



0.000 




0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 



1/[NH3] (mM-1) 






Fig. 3.5B plot of the reciprocal- fixed variable 
substrate NH3 versus slope (plus) and intercept (cross) from 
Fig . 3.5A. 



106 



0.012- 



0.010- 



0.008 



1 /V J.006- 



0.004- 



0.002 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[Asp] (mM-1) 



Fig. 3.6A Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of NH3 . Each initial velocity is the average 
of two experiments. The L-aspartate concentrations were 0.2, 
0.3, 0.4, 0.5 and 0.7 mM at various fixed concentrations of 
NH3: (lower triangle) 3.0 mM, (cross) 6.0 mM, (square) 12.0 
mM, (open circle) 24.0 mM, (plus) 36.0 mM. The concentrations 
of ATP and MgCl2 were 1 and 3 mM, respectively. 7.4 ug of 
enzyme per assay was used. 






107 



Slope & 
Intercept 



o.ooe- 



0.006- 



0.004- 



0.002- 



o.ooe 




0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 

1/[NH3] (mM-1) 



Fig. 3.6B plot of the reciprocal- fixed variable 
substrate NH3 versus slope (plus) and intercept (cross) from 
Fig. 3.6A. 



108 



1/v 



0.06 - 



0.05 - 



0.04 - 



0.03 - 



0.02 



0.01 - 



0.00 




Fig - 3 - 7 Double-reciprocal plot of initial velocity 
versus L-glutamme concentration at fixed concentration of 
LGH Each initial velocity is the average of two experiments 
ine L-glutamme concentrations were 0.21, 0.3, 05 07 09 
and 1.1 mM at various fixed concentrations of LGH: ' (plus) 
0.0, n(M, (cross) 0.08 mM, (square) 0.5 mM, (open circle) 1 
mM, (lower triangle) 3.0 mM. The concentrations of ATP, L- 
aspartate and MgCl 2 were 1, 1 and 3 mM, respectively. 7.4 ug 
of enzyme per assay was used. 






109 



1/v 



0.012-1 



0.010- 



0.008 



0.006- 



0.004- 



0.002 




0.000 







0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fi 9- 3 -8 Double-reciprocal plot of initial velocity 
versus ATP concentration at fixed concentration of L- 
glutamine (plus) and LGH (cross) (0.2 mM) . Each initial 
velocity is the average of two experiments. The ATP 
concentrations were 0.2, 0.3, 0.5 and 0.7 mM. The 
concentrations of L-aspartate and MgCl2 were 1 and 3 mM, 
respectively. 7.4 fig of enzyme per assay was used. 



110 



1/v 



0.008- 



0.006 



0.004- 



0.002 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[Asp] (mM-1) 



Fi 9- 3 - 9 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of ATP. Each initial velocity is the average 
of two experiments. The L-aspartate concentrations were 0.2, 
0.3, 0.5, 0.7, and 0.9 mM at various fixed concentrations of 
ATP: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, (open 
circle) 0.7 mM. The concentrations of L-glutamine and MgCl2 
were maintained at 20 and 3 mM, respectively. 7.4 ug of 
enzyme per assay was used. 



Ill 



0.006- 



1/V 



0.004- 



0.002 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[Gln] (mM-1) 



Fig. 3.10 Double-reciprocal plot of initial velocity 
versus L-glutamine concentration at various concentrations of 
ATP Each initial velocity is the average of two experiments, 
ine L-glutamine concentrations were 0.1, 0.2, 0.4, 0.5 and 
0.7 mM at various fixed concentrations of ATP: (plus) 1 mM 
(cross) 0.2 mM, (open circle) 0.3 mM, (square) 0.5 mM, (lower 
triangle) 0.5 mM, (eye) 0.7 mM. The concentrations of L- 
aspartate and MgCl 2 were 2 and 3 mM, respectively. 7.4 ug of 
enzyme per assay was used. 



112 



[Glu]/[PPI] 




8 10 12 14 16 18 20 

[Gin] mM 



Fig. 3.11 The ratio of L-glutamate produced/PPi produced 
versus concentration of L-glutamine. The assay mixtures (100 
111) contained 50 mM Tris-HCl (pH 8.0), 1 mM ATP, 1 mM L- 
aspartate, 3 mM MgCl2 and varying concentration of L- 
glutamine (0.1-20.0 mM) . The concentrations of L-glutamate 
and PPi were measured, as described under Materials and 
Methods. Each initial velocity is the average of two 
experiments . 









113 



1/v 



0.020 



0.015 



0.010- 



0.005- 



0.000 




0.00 0.05 0.10 0.15 0.20 

1/[NH3] (mM-1) 



0.25 0.30 0.35 



Fig. 3.12 Double-reciprocal plot of initial velocity 
versus NH3 concentration at various fixed concentrations of 
L-asparagine. Each initial velocity is the average of two 
experiments. The NH3 concentrations were 3.0, 6.0, 9.0, 12.0, 
and 15.0 mM at various fixed concentrations of L-asparagine:' 
(plus) 0.0 mM, (cross) 0.025 mM, (lower triangle) 0.05 mM, 
(eye) 0.075 mM, (square) 0.10, (open circle) 0.15. The 
concentrations of L-aspartate, ATP and MgCl2 were 1, 1, and 3 
mM, respectively. 7.4 ug of enzyme per assay was used. 









114 



1/v 



0.020- 



0.015- 



0.010- 



0.005 



0.000 




2 3 4 5 6 

1/[ATP] (mM-1) 



Fig. 3.13 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-asparagine. Each initial velocity is the average of two 
experiments. The ATP concentrations were 0.1, 0.3, 0.3, 0.4, 
and 0.5 mM at various fixed concentrations of L-asparagine:' 
(plus) 0.0 mM, (cross) 0.05 mM, (square) 0.10 mM, (open 
circle) 0.15 mM. The concentrations of L-aspartate, NH3 and 
MgCl2 were 1, 50, and 3 mM, respectively. 7.4 ug of enzyme 
per assay was used. 



115 



1/v 



0.015- 



0.010- 



0.005- 



0.000 




1/[Asp] (mM-1) 



Fig. 3.14 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-asparagine. Each initial velocity is the 
average of two experiments. The L-aspartate concentrations 
were 0.1, 0.3, 0.5, 0.7, and 0.9 mM at various fixed 
concentrations of L-asparagine: (plus) 0.0 mM, (cross) 025 
mM, (square) 0.05 mM, (open circle) 0.075 mM, (lower 
triangle) 0.10 mM, (eye) 0.15 mM. The concentrations of ATP, 
NH3 and MgCl2 were 1, 50, and 3 mM, respectively. 7.4 ug of' 
enzyme per assay was used. 



116 



1/v 



0.020- 



0.015- 



0.010 



0.005 




3456789 10 

1/[ATP] (mM-1) 



Fig. 3.15 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
^ P \^ Ch initial velocity is the average of two experiments. 
The ATP concentrations were 0.1, 0.3, 0.5, 0.7, and 0.9 mM 
at various fixed concentrations of AMP: (plus) 0.0 mM, 
(cross) 3.0 mM, (square) 6.0 mM, (open circle) 9.0 W (eye) 
12.0 mM. The concentrations of L-aspartate and NH 3 were 1 and 
50 mM, respectively. MgCl 2 concentration was as to provide 1 
mM excess above ATP and AMP. 7.4 ^g of enzyme per assay was 
used. 



117 



1/V 



0.020 



0.015 



0.010- 



0.005- 



0.000 




0123456789 10 

1/[Asp] (mM-1) 



Fig. 3.16 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of AMP. Each initial velocity is the average 
of two experiments. The ATP concentrations were 0.1, 0.3, 
0.5, 0.7, and 0.9 mM at various fixed concentrations of AMP: 
(plus) 0.0 mM, (cross) 3.0 mM, (square) 6.0 mM, (open circle) 
9.0 mM. The concentrations of ATP and NH3 were 1 and 50 mM, 
respectively. MgCl2 concentration was as to provide 1 mM 

excess above ATP and AMP. 7.4 ug of enzyme per assay was 
used. 



1/v 



0.020- 



0.015- 



0.010 



0.005 



0.000 




0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 

1/[NH3] (mM-1) 



Fig. 3.17 Double-reciprocal plot of initial velocity 
versus NH3 concentration at various fixed concentrations of 
AMP. Each initial velocity is the average of two experiments. 
The NH3 concentrations were 3.0, 6.0, 9.0, 12.0, and 15 mM at 
various fixed concentrations of AMP: (plus) 0.0 mM, (cross) 
3.0 mM, (square) 6.0 mM, (open circle) 9.0 mM. The 
concentrations of ATP and L-aspartate were 1 and 1 mM, 
respectively. MgCl2 concentration was as to provide 1 mM 
excess above ATP and AMP. 7.4 ug of enzyme per assay was 
used. 



1/v 




0.005 



0.000 



1/[Gln] (mM-1) 



Fig. 3.18 Double-reciprocal plot of initial velocity 
versus L-glutamine concentration at various fixed 
concentrations of L-asparagine. Each initial velocity is the 
average of two experiments. The L-glutamine concentrations 
were 0.1, 0.3, .0.5, 0.7 and 0.9 mM at various fixed 
concentrations of L-asparagine: (plus) 0.0 mM, (cross) 05 
mM, (square) 0.10 mM, (open circle) 0.15 mM. The 
concentrations of L-aspartate, ATP and MgCl2 were 1, 1, and 3 
mM. 7.4 ng of enzyme per assay was used. 



120 



0.025- 



0.020- 



1 /V 5.015 



0.010- 



0.005- 






0.000 




2 4 6 8 10 12 14 16 18 20 

1/[ATP] (mM-1) 



Fig. 3.19 Double-reciprocal plot of initial velocity 
versus ATP at various fixed concentrations of L-asparagine. 
Each initial velocity is the average of two experiments . The 
ATP concentrations were 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM 
at various fixed concentrations of L-asparagine: (plus) 0.0 
mM, (cross) 0.05 mM, (square) 0.10 mM, (open circle) 0.15 mM, 
(lower triangle) 0.20 mM. The concentrations of L-aspartate, 
L-glutamine and MgCl2 were 1, 1, and 3 mM. 7.4 ug of enzyme 
per assay was used. 



121 



0.015- 



0.010- 



1/V 



0.005- 



0.000 




1/[Asp] (mM-1) 



Fig. 3.20 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-asparagine . Each initial velocity is the 
average of two experiments. The L-aspartate concentrations 
were 0.1, 0.3, 0.5, 0.7 and 0.9 mM at various fixed 
concentrations of L-asparagine: (plus) 0.0 mM, (cross) 0.05 
mM, (square) 0.10 mM, (open circle) 0.15 mM, (lower triangle) 
0.20 mM. The concentrations of L-glutamine, ATP and MgCl2 
were 1, 1, and 3 mM. 7.4 \ig of enzyme per assay was used. 



1/v 







1/[ATP] (mM-1) 



Fig. 3.21 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
PPi. Each initial velocity is the average of three 
experiments. The ATP concentrations were 0.1, 0.3, 0.5, 0.7 
and 0.9 mM at various fixed concentrations of PPi: (plus) 0.0 
mM, (cross) 0.20 mM, (open circle) 0.60 mM, (upper triangle) 
0.80 mM. The concentrations of L-glutamine and L-aspartate 
were 1 and 1 mM. MgCl2 concentration was as to provide 1 mM 
excess above ATP and PPi, 
used. 



7.4 ng of enzyme per assay was 



1/v 



0.4 



0.3 - 



0.2 - 



0.1 - 



0.0 




01 23456789 10 

1/[Asp] (mM-1) 



Fig. 3.22 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of ppj. . Each initial velocity is the average 
of three experiments. The L-aspartate concentrations were 
0.1, 0.3, 0.5, 0.7 and 0.9 mM at various fixed concentrations 
of PPi: (plus) 0.0 mM, (cross) 0.20 mM, (square) 4 mM 
(open circle) 0.60 mM. The concentrations of L-glutamine and 
ATP were 1 and 1 mM. MgCl 2 concentration was as to provide 1 

mM excess above ATP and PPi. 7 . 4 ug of enzyme per assay was 
used. 



124 



1/v 



0.006- 



0.004 



0.002 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fig. 3.23 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-glutamate. Each initial velocity is the average of two 
experiments. The ATP concentrations were 0.1, 0.2, 0.3, 0.4, 
0.5, and 0.7 at various fixed concentrations of L-glutamate: 
(plus) 0.0 mM, (cross) 15.0 mM, (square) 20.0 mM, (open 
circle) 40.0 mM. The concentrations of L-glutamine and L- 
aspartate were 1 and 1 mM, respectively. MgCl2 concentration 
was kept constant (3 mM) . 7.4 [lg of enzyme per assay was 
used. 



125 



0.020- 



0.015 



1/v 



0.010- 



0.005- 



0.000 




1/[Asp] (mM-1) 



Fig. 3.24 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-glutamate. Each initial velocity is the 
average of two experiments. The L-aspartate concentrations 
were 0.1, 0.2, 0.3, 0.4, 0.5, and 0.7 mM at various fixed 
concentrations of L-glutamate: (plus) 0.0 mM, (cross) 5.0, 
(square) 10.0, (open circle) 15.0 mM, (upper triangle) 20.0 
mM, (eye) 30.0 mM. The concentrations of L-glutamine and ATP 
were 1 and 1 mM, respectively. MgCl2 concentration was kept 
constant (3 mM) . 7.4 ng of enzyme per assay was used. 



126 



0.020 



0.015 



1/V 




Fig. 3.25 Double-reciprocal plot of initial velocity 
versus L-glutamine concentration at various fixed 
concentrations of L-glutamate. Each initial velocity is the 
average of two experiments. The L-glutamine concentrations 
were 0.1, 0.2, 0.3, 0.4, 0.5, and . 7 at various fixed 
concentrations of L-glutamate: (plus) 0.0 mM, (cross) 10.0, 
(square) 20.0, (open circle) 30.0 mM, (A) 40.0 mM. The 
concentrations of L-aspartate and ATP were 1 and 1 mM, 
respectively. MgCl2 concentration was kept constant (3 mM) , 
7.4 ug of enzyme per assay was used. 



127 



0.008- 



0.006 



1/v 



0.004- 



0.002- 



0.000 




1.0 1.5 2.0 

1/[Gln] (mM-1) 



Fig. 3.26 Double-reciprocal plot of initial velocity 
versus L-glutamine concentration at various fixed 
concentrations of AMP. Each initial velocity is the average 
of two experiments. The L-glutamine concentrations were 0.1, 
0.3, 0.5, 0.7 and 0.9 mM at various fixed concentrations of 
AMP: (plus) 0.0 mM, (cross) 3.0, (square) 6.0, (open circle) 
9.0 mM, (upper triangle) 12.0 mM. The concentrations of L- 
aspartate and ATP were 1 and 1 mM, respectively. MgCl2 
concentration was as to provide 1 mM excess above ATP and 
AMP. 7.4 (ig of enzyme per assay was used. 



128 



1/V 



0.015- 



0.010 



0.005- 



0.000 




1/[Asp] (mM-1) 



Fig. 3.27 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of AMP. Each initial velocity is the average 
of two experiments. The L-aspartate concentrations were 0.1, 
0.3, 0.5, 0.7 and 0.9 mM at various fixed concentrations of 
AMP: (plus) 0.0 mM, (cross) 3.0, (square) 6.0, (open circle) 
9.0 mM, (upper triangle) 12.0 mM. The concentrations of L- 
glutamine and ATP were 1 and 1 mM. MgCl2 concentration was as 

to provide 1 mM excess above ATP and AMP. 7.4 [ig of enzyme 
per assay was used. 



129 



1/v 



0.010- 



0.008- 



0.006- 



0.004 



0.002- 



0.000 




01 23456789 10 

1/[ATP] (mM-1) 



Fig. 3.28 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
AMP. Each initial velocity is the average of two experiments 
The ATP concentrations were 0.1, 0.2, 0.3, 0.4, 0.5, mMat 
various fixed concentrations of AMP: (plus) 0.0 mM, (cross) 
3.0, (square) 6.0, (open circle) 12.0 mM. The concentrations 
of L-aspartate and L-glutamine were 1 and 1 mM, respectively 
MgCl2 concentration was as to provide 1 mM excess above ATP 
and AMP. 7.4 ug of enzyme per assay was used. 



130 



1/v 



0.010 



0.008 



n.006- 



0.004- 



0.002- 



0.000 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[Asp] (mM-1) 



Fig. 3.29 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-glutamine in the presence of L-glutamate 
(50 mM) . Each initial velocity is the average of two 
experiments. The L-aspartate concentrations were 0.1, 0.2, 
0.3, 0.5, and 0.7 mM at various fixed concentrations of L- 
glutamine: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.4 mM, (lower triangle) 0.7 mM. The 
concentrations of ATP and MgCl2 were 1 and 3 mM, 
respectively. 7.4 |lg of enzyme per assay was used. 



131 




0.10 



0.05 - 



0.00 



0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 



1/[Asp] (mM-1) 



Fig. 3.30 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at various fixed 
concentrations of L-glutamine in the presence of PPi (0 4 
m). Each initial velocity is the average of two experiments, 
ine L-aspartate concentrations were 0.2, 0.3, 0.4, 0.5, 0.7 
mM, 0.9 and 1.1 mM at various fixed concentrations of L- 
glutamme: (plus) 0.2 mM, (cross) 0.3 mM, (square) 4 mM 
The concentrations of ATP and MgCl 2 were 1 and 3 mM, ' 
respectively. 7.4 p.g of enzyme per assay was 



132 



0.010- 



0.008- 



0.006- 



1/V 



0.004 



0.002- 



0.000 




0.00 0.02 0.04 0.06 0.08 0.10 0.12 

[Asn] (mM) 



Fig. 3.31 Double inhibition studies plot of L-asparagine 
concentration versus reciprocal initial velocity at various 
fixed concentrations of AMP. Each initial velocity is the 
average of two experiments. The L-asparagine concentrations 
were 0, 0.025, 0.05, 0.075, 0.1, and 0.125 mM at various 
fixed concentrations of AMP: (plus) 0.0, (cross) 3.0 mM, 
(square) 9.0 mM, (open circle) 18.0 mM. The ATP, L-aspartate, 
and L-glutamine were all kept constant and subsaturating (1 
mM) . MgCl2 concentration was as to provide 1 mM in excess of 
total nucleotide concentrations. 7.4 ug of enzyme per assay 
was used. 









133 



0.005- 



1/V 



0.004 



0.003- 




10 15 20 25 30 35 

[Glu] (mM) 



Fig. 3.32 Double inhibition studies plot of L-glutamate 
concentration versus reciprocal initial velocity at various 
fixed concentrations of AMP. Each initial velocity is the 
average of two experiments. The L-glutamate concentrations 
were 0, 5.0, 10.0, 20.0, 30.0, and 4 0.0 mM at various fixed 
concentrations of AMP: (plus) 0.0, (cross) 3.0 mM; (square) 
9.0 mM, (open circle) 18.0 mM. The ATP, L-aspartate , and L- 
glutamine were all kept constant and subsaturating. MgCl2 
concentration was as to provide 1 mM in excess of total 
nucleotide concentrations. 7.4 |ig of enzyme per assay was 
used. 



134 



1/V 




10 15 20 25 30 35 40 

[Glu] (mM) 



Fig. 3.33 Double inhibition studies plot of L-glutamate 
concentration versus reciprocal initial velocity at various 
fixed concentrations of L-asparagine. Each initial velocity 
is the average of two experiments. The L-glutamate 
concentrations were 0, 5.0, 10.0, 20.0, 30.0, and 40.0 mM at 
various fixed concentrations of L-asparagine: (plus) 0, 
(cross) 0.025 mM, (square) 0.05 mM, (open circle) 0.075 mM, 
and (upper triangle) 0.1 mM. The ATP , L-aspartate, and L- 
glutamine were all kept constant and subsaturating. MgCl2 

concentration was maintained at 3 mM. 7.4 ug of enzyme per 
assay was used. 



135 



1/V 



0.030- 



0.025- 



0.020- 



0.015- 



0.010 



0.005- 



0.000 




01 23456789 10 



1/[Asp] (mM-1) 



Fig. 3.34 Double-reciprocal plot of initial velocity 
versus L-aspartate concentration at fixed varied 
concentrations of L-glutamate and AMP in a constant ratio 
( [L-glutamate] - 5 [AMP] ) . Each initial velocity is the 
average of two experiments. The L-aspartate concentrations 
were 0.1,, 0.3, 0.5, 0.7 and 0.9 mM at fixed varied 
concentrations of L-glutamate: (plus) 0.0 mM, (cross) 10.0 
mM, (square) 50.0 mM, (open circle) 70.0 mM, and of AMP: 
(plus) 0.0 mM, (cross) 2.0 mM, (square) 10.0 mM, (open 
circle) 14.0 mM at a fixed ration (5). The concentrations of 
ATP and L-glutamine we re 1 and 1 mM, respectively. MgCl2 
concentration was as to provide 1 mM in excess of total 
nucleotide concentrations. 7.4 p.g of enzyme per assay was 
used. 



136 



1/v 



9.8 - 
9.7 
9.6 
9.5 
9.4 - 
9.3 
9.2 
9.1 
9.0 - 

8.9 - 
8.8 - 




3456789 10 

1/[ATP] (mM-1) 



Fig. 3.35. Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 25, derived to fit the complex mechanism 
(Scheme D) The ATP concentrations were assumed to be 0.1, 
u.A U.J, 0.5, and 0.7 mM at various fixed concentrations of 
L-aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 3 mM 
(open circle) 0.5 mM, (lower triangle) 0.7 mM. The 
concentration of L-glutamine was maintained at 1 mM All k«s ' 
were assumed to be equal to 1 . ' 



137 



200 



150 



1/V 



100 - 



50 




23456789 10 

1/[ATP] (mM-1) 



Fig. 3.36. Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 25, derived to fit the complex mechanism 
(Scheme D) . The ATP concentrations were assumed to be 0.1, 
0.2, 0.3, 0.5, and 0.7 mM at various fixed concentrations of 
L-aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.5 mM, (lower triangle) 0.7 mM. The 
concentration of L-glutamine was maintained at 1 mM. All ks ' 
with exception of kl5 were assumed to be equal to 1. kl5 (k on 
for L-glutamine for the side reaction) was assumed to be 
equal to . 



138 



1/V 



26 

24 

22 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 






I I I I I I I I I | 
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fig. 3.37. Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 

VK^ ^f^S? 25 ' derived ^ fit the complex mechanism 
(Scheme D) . The ATP concentrations were assumed to be 2 
U.J, 0.5, and 0.7 mM at various fixed concentrations of L- 
aspartate: (plus) 0.2 mM, (cross) 0.3 mM, (square) 5 mM 
(upper triangle) 0.7 mM. The concentration of L-glutamine'was 
maintained at 20 mM. All ks ■ with exception of k?5 were 
assumed to be equal to 1. kl5 (k on for L-glutamine for the 
side reaction) was assumed to be equal to 0.004. 









139 



90 -i 



1/V 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 



1/[ATP] (mM-1) 



Fig. 3.38 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 25, derived to fit the complex mechanism 
(Scheme D) . The ATP concentrations were assumed to be 0.2, 
0.3, 0.5, and 0.7 mM at various fixed concentrations of L- 
aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.5 mM, (lower triangle) 0.7 mM. The 
concentration of L-glutamine was maintained at 5 mM. 
with exception of kl5 were assumed to be equal to 1. 
for L-glutamine for the side reaction) was assumed to 
equal to 0.002. 



All 

kl5 

be 



ks' 
(*on 












140 



1/v 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 



Fig. 3.39 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 25, derived to fit the complex mechanism 
(Scheme D) . The ATP concentrations were assumed to be 0.2, 
0.3, 0.5, and 0.7 mM at various fixed concentrations of L- 
aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.5 mM, (lower triangle) 0.7 mM. The 
concentration of L-glutamine was maintained at 10 mM. All ks ' 
with exception of kl5 were assumed to be equal to 1. kl5 (k n 
for L-glutamine for the side reaction) was assumed to be 
equal to 0.002. 



141 



1/v 




0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 

1/[ATP] (mM-1) 






Fig. 3.40 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 25, derived to fit the complex mechanism 
(Scheme D) . The ATP concentrations were assumed to be 0.2, 
0.3, 0.5, and 0.7 mM at various fixed concentrations of L- 
aspartate: (plus) 0.1 mM, (cross) 0.2 mM, (square) 0.3 mM, 
(open circle) 0.5 mM, (lower triangle) 0.7 mM. The 
concentration of L-glutamine was maintained at 20 mM. All ks ' 
with exception of kl5 were assumed to be equal to 1. kl5 (k on 
for L-glutamine for the side reaction) was assumed to be 
equal to 0.002. 



142 



0.0020- 



1/V 



0.0015- 



0.0010- 




3456789 10 

1/[ATP] (mM-1) 



Fig. 3.41 Double-reciprocal plot of initial velocity 
versus ATP concentration at various fixed concentrations of 
L-aspartate. This plot was generated by the Quatro program 
using equation 24, derived to fit the simple mechanism 
(Scheme A). The ATP concentrations were assumed to be 0.1, 
0.2, 0.3, 0.5, 0.7, 0.9 and 1.1 mM at various fixed 
concentrations of L-aspartate: (plus) 0.1 mM, (cross) 0.2 mM, 
(square) 0.3 mM, (open circle) 0.5 mM. The concentration of 
L-glutamine was maintained at 1 mM. All ks ' with exception of 
k7 were assumed to be equal to 1 . k7 (k D ff for L-glutamate) 
was assumed to be equal to -0.02. 



143 



Table 3.1 Inhibition patterns for ASB obtained with 0- 
methyl aspartate, AMP-PNP and L-glutamic acid y-methyl ester 
with respect to L-aspartate, ATP and L-glutamine. 



Substrates 


Analogs 


pattern 


Kis (niM) 


Kii (mM) 




AMP-PNP 








ATP 




C 


0.91 


- 


L-aspartate 




NC 


5.2 


4.5 


L-glutamine 


p-methyl 
aspartate 


uc 




3.2 


ATP 




NC 


8.15 


15 


L-aspartate 




C 


18 


>50 


L-glutamine 


L-glutamic 


NC 


93 


16.5 




acid y-methyl 










ester 








ATP 




U 


- 


10.4 


L-aspartate 




UC/NC 


- 


9 


L-glutamine 




C 


6.6 


3.2 



The velocities were measured spectrophotometrically as 
described before. Each initial velocity is the average result 
of two parallel experiments. The assay mixture contained the 
following components: 100 mM Tris-HCl (pH 8.0), 5 . mM MgCl2 
and varying amounts of ATP (0.05-0.6 mM) , L-aspartate (0.1- 
1.1 mM) , L-glutamine (0.1-0.9 mM) . Analogs were varied from 
to 3.0 mM (^-methyl aspartate), to 4.0 mM (AMP-PNP) and 
to 5.0 mM (L-glutamic acid y-methyl ester). The volume of the 
total reaction mixture was kept at 160 (J.1 . All reactions were 
carried out at 37°C, using 5.0 |ig of enzyme per reaction. a C 
= competitive, NC = noncompetitive, and UC = uncompetitive 



144 



Table 3.2 Product inhibition data for ammonia-dependent 
reaction of ASB. 









Ki (mM) 




Varied a 


Inhibitor 


Inhibition 


Slope Intercept 


Substrate 




Pattern 






NH 3 


AMP 


NC 


15 ± 0.002 


7 ± 0.001 


L-Asp 


AMP 


NC 


8 ± 0.001 


11 ± 0.001 


ATP 


AMP 


NC 


3 ± 0.001 


12 ± 0.001 


NH 3 


L-Asn 


C 


0.08 ± 0.025 






L-Asp 


L-Asn 


NC 


0.26 ± 0.002 


0.7 ± 0.001 


ATP 


L-Asn 


NC 


0.19 ± 0.002 


0.15 ± 0.001 


he velocities were m 


easured spe 


ctrophotometri 


callv as 



described under Materials and Methods. 
a Other substrates are at saturating constant levels 
C, competitive; NC, noncompetitive 



145 



Table 3.3 Product inhibition data for glutamine- 
dependent reaction of ASB. 



Varied a 


Inhibitor 


Inhibition 


Substrate 




Pattern 


L-Gln 


L-Asn 


C 


L-Asp 


L-Asn 


NC 


ATP 


L-Asn 


NC 


L-Asp 


PP1 


C 


ATP 


PP1 


C 


L-Gln 


AMP 


NC 


L-Asp 


AMP 


NC 


ATP 


AMP 


NC 


L-Gln 


L-Glu 


NC 


L-Asp 


L-Glu 


C 


ATP 


L-Glu 


NC 



Ki (mM) 
Slope Intercept 



0.015 ± 0.003 

0.09 ± 0.001 0.25 ± 0.001 

0.27 ± 0.001 0.17 ± 0.002 

0.39 ± 0.01 

0.05 ± 0.002 

14 ± 0.001 10 ± 0.001 

9 ± 0.001 47 ± 0.001 

5 ± 0.001 22 ± 0.001 

52 ± 0.001 133 ± 0.001 

23 ± 0.001 

47 ± 0.002 47 ± 0.001 



The velocities were measured spectrophotometrically as 
described under Materials and Methods. 
a Other substrates are at saturating constant levels. 
C, competitive; NC, noncompetitive 









CHAPTER 4 
EFFECT OF TEMPERATURE ON THE ASPARAGINE SYNTHETASE B 



Introduction 

The glutamine and ammonia-dependent reactions catalyzed 
by E. coli asparagine synthetase B were studied extensively 
in the steady-state (Chapters 2 and 3). The reactions were 
found to occur by an ordered ternary ping-pong mechanism. In 
this chapter, we present the steady state kinetics of E. coli 
ASB over a range of temperatures. Arrhenius plots were used 
to detect any changes in the rate limiting steps for the 
enzyme catalyzed reactions. We wished to determine whether a 
change in slope of the plot can be detected in either 
reaction, and whether such a change can be different with 
glutamine- and ammonia- dependent reactions. In other words, 
is the rate limiting step different for the ammonia- and 
glutamine-dependent reactions? 

Materials and Methods 

Chemicals and Reagents 

Trichloroacetic acid (TCA) was purchased from Fisher 
Scientific (Orlando, PL) . The pyrophosphate reagent for 
following PPi production, MgCl2, ATP, L-aspartate, L- 



146 



147 



glutamine, ammonium acetate, Tris (hydroxymethyl) aminomethane 
(Tris-HCl) , were all purchased from Sigma. Dithiothreitol 
(DTT) was obtained from Promega Corporation (Madison, 

Wisconsin) . 

Expression of t he Protein and Purification 

Protein expression, cell culture and enzyme purification 
was carried out as described before (Chapter 2) . 

Protein Concentration Determination 

Protein concentration was measured using Bio-Rad Protein 
Assay (Bradford, 1976) . Mouse immunoglobulin G was used to 
obtain a standard curve. 

Enzyme Assays 

The effect of temperature on the kinetic parameters was 
investigated with experiments conducted at 0.6, 5, 10, 15, 
20, 25, 30, 35, and 40°C. The initial rate assays were 
carried out at pH 8.0 in 50 mM Tris-HCl. The pH was adjusted 
at each temperature. The concentration of substrates when 
varied were 0.1-1.1 mM for ATP, L-aspartate and L-glutamine 
and 5-40 mM for ammonium acetate. The concentration of the 
substrates when held constant and saturating were 5 mM, 10 
mM, 10 mM and 50 mM for ATP, L-aspartate, L-glutamine and 
ammonia, respectively. MgCl2 concentration was keep constant 
(8 mM). A typical assay was performed as follows. To a 80 [ll 



148 



reaction mixture containing all the substrates minus the 
enzyme, preincubated for 3.5 min at desired temperature, 20 
|il of ASB (7.4 ng) was added to start the reaction. Reactions 
were terminated by addition of 20% TCA (15 jj,l) . PPi 
production was measured by modifying the continuous 
spectrophotometric assay (0' Brian, 1976) to an end point 
assay. In this case, 385 (il of the coupling buffer (50 mM 
imidazole, pH not adjusted, and 20 (il of pyrophosphate 
reagent, which was originally reconstituted in 1 ml of 
ddH20) , was added to the reaction mixtures, following TCA 
termination, and incubated at room temperature for 30 min. 
The absorbance of the resulting solution was measured at 340 
nm, and the amount of pyrophosphate produced in the reaction 
determined from a standard curve. 

The exact temperature in the reaction mixture was 
monitored by a thermometer during the experiment. At the 
highest temperature (40°C) the enzyme was stable during the 
assay period. All studies were done in duplicates, and Vmax 
and Km were obtained from least-square analyses of the double 
reciprocal plots of 1/V vs 1/ [substrate] at each temperature. 

Thermodynamics of Activation 

Activation energies, entropic and enthalpic components 
at a given temperature were calculated from slopes of plots 
of log velocity versus 1/T, using the following equations: 

AG* = RT ln(k B T/h) - RTln(k) 



149 



k = Aexp[-Ea/RT] 
Ea = Ah* + rt 

AG* = Ah* - tAs* 
where K B is Boltzmann's constant, h is Planck's constant, k is 



the forward rate constant, Ea is the Arrhenius activati 



on 



energy, A is a constant. The free energy of activation, AG*, 

is directly related to the reaction rate. The enthalpy of 
activation, Ah* is a measure of the energy barrier that the 
reacting molecules must overcome. As* is the entropy of 
activation. 

Results and Discussion 

To investigate the effect of temperature upon E. coli 
ASB, initial rates were measured at different temperatures 
(0-40°C) . The Log of maximum steady state velocity was 
plotted against 1/T. Our results are shown in Figures 4.1- 
4.3 for the ammonia-dependent reaction (varying NH3 , ATP and 
L-aspartate, respectively) and Figure 4.4 for the glutamine- 
dependent reactions (varying L-glutamine) . In all cases, 
regardless of what the varied substrate was, straight lines 
were obtained which suggested that the velocity is controlled 
by one rate constant over the temperature range. This can 
also suggest an overall structural change in the protein that 
is common to both reactions. From the plot of Log Vmax versus 
temperature (1/T), varying NH3 , the Arrhenius activation 
energy was calculated to be 63.3 kJ/mol. The energetic 



150 



parameters for the ammonia-dependent reaction, varying NH3 , 

were calculated to be (at 37°C) 132 kJ/mol, 61 kJ/mol, and 
-72 kJ/mol for AG*, Ah*, TAS*, respectively. When L-aspartate 

and ATP were the varied substrates, the energetic parameters 
calculated from the plots were about the same (not shown) . 
The Arrhenius activation energy for the glutamine-dependent 
reaction, varying L-glutamine, (at 37°C) was found to be 44.2 
kJ/mol, and AG*, AH* and TAS* were found to be 114 kJ/mol, 42 

kJ/mol and -72 kJ/mol, respectively. Similar values were 
obtained when ATP and L-aspartate were the varied substrates 
(not shown) . As the data show, the entropies of activation 
are essentially the same for both reactions. On the other 
hand, the AG* is lower for the glutamine-dependent reaction 

than for the ammonia-dependent reaction which is essentially 
enthalpic in nature. This strongly suggests that at the 
transition state, the electrostatic interaction is more 
favored in the presence of L-glutamine than in the presence 
of NH3 . It is possible that the ASB rigidly holds the L- 
glutamine in place so that it can attack the aspartyl-AMP 
intermediate as soon as it is formed. It also possible that 
ASB by polarizing the glutamine bonds, makes it a better 
nucleophilic group, lowering the activation energy (Hammes, 
1964) . 

The Km values for ATP, L-aspartate and L-glutamine did 
not change with temperature for ammonia- and glutamine- 
dependent reactions. This suggests that the energy in AG° is 
from TAS° (100%) . However, the Km is the sum of rate 



151 



constants (Chapter 2), and no information regarding the 
effect of temperature on these rate constants is available at 
this time. 

Based on the data presented in Chapter 1 and 2, the 
glutamine- and ammonia-dependent reactions were found to 
occur by an ordered ternary ping-pong mechanism. According to 
the mechanism, ATP and L-aspartate bind first, forming 
aspartyl-AMP. This is followed by release of PPi (P) and 
addition of NH3 or L-glutamine for ammonia- and glutamine- 
dependent reaction, respectively. Basically, the kinetic 
mechanism up to the formation of aspartyl-AMP is the same for 
the two reactions. This suggests that activation energy 
should be the same for the two reactions up to the formation 
of aspartyl-AMP. However, our data showed that activation 
energy was different for ammonia-and glutamine-dependent 
reactions. This suggests the activation energy that we have 
measured corresponds to a step following the aspartyl-AMP 
formation; that is the addition of NH3 or L-glutamine. The 
fact that activation energy was lower for glutamine-dependent 
reaction suggests that L-glutamine binds the enzyme which 
would in turn polarize the glutamine bonds, making it a 
better nucleophilic group. This would then attack aspartyl- 
AMP. NH3, however, which is a better nucleophile, cannot bind 
the enzyme as well, therefore having a higher activation 
energy. 



152 



x 
a 

E 
> 

O) 

o 



10 
9. 
8. 
7. 
6. 
5- 
4 

3. 
2. 
1. 




I I I I | I I 

0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037 

1/T 



Fig. 4.1. Arrhenius plot of the Vmax values for the 
ammonia-dependent reaction, varying NH3 concentration- The 
initial rate assays were carried out at pH 8.0 in 50 mM Tris- 
HC1. The NH3 concentration was varied (5-40 mM) , while the 
concentrations of L-aspartate, ATP and MgCl2 were maintained 
at 10, 5 and 8 mM, respectively. 7.4 |ig of enzyme per assay 
was used. All studies were done in duplicate, and the data 
were analyzed by the double reciprocal of 1/V vs 1/[NH3]. 
Velocities, used in the Arrhenius plot, were reported as 
nmole of L-asparagine produced per minute per milligram of 
protein. 












153 



I 



4.0_ 

3.5. 

3.0. 

2.5. 

2.0. 

1.5. 

1.0. 

0.5. 

0.0. 



1 1 1 1 1 1 1 

0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037 

1/T 



Fig- 4.2. Arrhenius plot of the Vmax values for the 
ammonia-dependent reaction, varying ATP concentration. The 
initial rate assays were carried out at pH 8.0 in 50 mM Tris- 
HC1. The ATP concentration was varied (0.1.1 mM) , while the 
concentrations of L-aspartate, L-glutamine and MgCl2 were 
maintained at 10, 10 and 8 mM, respectively. 7.4 (ig of enzyme 
per assay was used. All studies were done in duplicate, and 
the data were analyzed by the double reciprocal of 1/V vs 
1/[ATP]. Velocities, used in the Arrhenius plot, were 
reported as nmole of L-asparagine produced per minute per 
milligram of protein. 









154 



3 

E 

> 

O) 

o 




00530 ° 0031 00032 0.0033 0.0034 0.o!)3 5 0.o!)36 0.0037 



1/T 



Fig. 4.3. Arrhenius plot of the Vmax values for the 
ammonia-dependent reaction, varying L-aspartate 
concentration. The initial rate assays were carried out at pH 
8.0 in 50 mM Tris-HCl. The L-aspartate concentration was 
varied (0.1-1.1 mM) , while the concentrations of L-aspartate, 
ATP and MgCl2 were maintained at 10, 5 and 8 mM, 

respectively. 7.4 |ig of enzyme per assay was used. All 
studies were done in duplicate, and the data were analyzed by 
the double reciprocal of 1/v vs 1/ [L-aspartate] . Velocities, 
used in the Arrhenius plot, were reported as nmole of L- 
asparagine produced per minute per milligram of protein. 






155 




0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037 

1/T 



Fig. 4.4. Arrhenius plot of the Vmax values for the 
glutamine-dependent reaction, varying L-glutamine 
concentration. The initial rate assays were carried out at pH 
8.0 in 50 mM Tris-HCl. The L-glutamine concentration was 
varied (0.1-1.1 mM) , while the concentrations of L-aspartate, 
ATP and MgCl2 were maintained at 10, 5 and 8 mM, 

respectively. 7.4 p.g of enzyme per assay was used. All 
studies were done in duplicate, and the data were analyzed by 
the double reciprocal of 1/V vs 1/ [L-glutamine] . Velocities, 
used in Arrhenius plot, were reported as nmole of L- 
asparagine produced per minute per milligram of protein. 



156 



Table 4.1 Thermodynamic properties for ammonia- and 
glutamine-dependent reactions of ASB. 



ASB reactions 



1) NH 3 + L-Asp + ATP- 
L-Asn + AMP + PP^ 



2) L-gln + L-Asp + ATP 

L-Asn + AMP + PP^ + L-Glu 



AG* 

kJ mol" 1 , 
(37°C) 



AH* 

kJmol - !, 
(0.5-37°C) 



AS* 

Jmol-iR" 1 , 

(0.5-37°C) 



TAS* 

kJ mol - -'-, 
(37°C) 



132 ± 13 



61 ± 6.1 



-231 ± 23 



-72 ± 7 



114 ± 11 



42 ± 4 



-233 ±23 



-72 ± 7 



The initial rates were determined at pH 8.0 in 50 mM Tris-HCl 
at different assay temperatures (0.6-40°C), under conditions 
described in materials and methods. 



CHAPTER 5 
SUMMARY AND CONCLUSIONS 

The successful remission of certain types of cancers 

upon treatment with B. coli asparaginase as the 

chemotherapeutic agent has been correlated to a decreasing 

concentration of the amino acid L-asparagine. Such 

observations led to the suggestion that a highly specific and 

potent inhibitor of asparagine synthetase (AS) , the enzyme 

responsible for synthesis of L-asparagine, might be effective 

in treating tumors. Therefore, understanding the biosynthesis 

L-asparagine is important for the development of AS 

inhibitors that would otherwise decrease the availability of 

endogenous L-asparagine. To increase our knowledge of L- 

asparagine metabolism, the enzyme AS has been investigated 

from several different sources. Very little information is 

available regarding the chemical mechanism of AS. Previous 

studies have clearly indicated that the enzyme utilizes an 

aspartyl-AMP intermediate. Attempts to define the pathway by 

which the amide nitrogen is transferred from the L-glutamine 

to this activated complex in AS have been reported. To date, 

the most widely accepted mechanistic hypothesis involves the 

nucleophilic attack upon aspartyl-AMP by ammonia, implying 

that L-glutamine undergoes hydrolysis (Zalkin et al., 1989). 

Evidence for this hypothesis was provided by the alignment of 

the primary sequences of several glutamine-dependent 

157 



158 



amidotransf erases, including human AS, which revealed the 
presence of a conserved triad consisting of histidine (His- 
101), cysteine (Cys-1) and aspartic acid (Asp-29) residues 
(see Chapter 1) . Mutagenesis of these residues resulted in 
the loss of glutamine- but not ammonia-dependent activity, 
suggesting that Cys-1 participates in an amide hydrolysis 
reaction to release ammonia, and His and Asp act as general 
bases in the reaction. This hypothesis was later challenged 
by Richards and Schuster (1992) . Upon finding that E. coli 
ASB lacks the conserved His residue in the proposed catalytic 
triad but still exhibits similar specificity to human AS, 
they suggested a new proposal for the nitrogen transfer 
mechanism in which glutamine reacts directly with aspartyl- 
AMP to form an imide intermediate. The reason why this is so 
important is that if an imide intermediate is formed, it 
opens numerous possibilities for design of new, potent 
mechanism-based inhibitors. It was for this reason that the 
mechanism is important . 

The kinetic mechanism of E. coli ASB was studied to 
obtain information about the order of the binding of the 
substrates and release of products. This was done therefore, 
to eliminate possible intermediates in the mechanism. For 
example, if L-glutamate was released prior to the binding of 
either ATP or L-aspartate, the current mechanistic hypothesis 
involving the imide intermediate (Richards and Schuster, 
1992) would effectively be ruled out. 



159 



In chapter 2 of this work the kinetic mechanism of E. 
coli ASB was examined using isotope partitioning. When 
radioactive L-aspartate was used in the pulse in the presence 
or absence of L-glutamine (Table 2.1), very little L-Asp* 
(10%) was trapped as L-Asn*. However, when isotope 
partitioning experiments were done with ATP in addition to 
the L-Asp* in the pulse solution, 90% of E-Asp*-ATP was 
trapped as L-Asn* . The ability to trap radioactive L- 
aspartate when ATP was included in the pulse solution 
suggests that the E-Asp*-ATP complex is formed in a 
catalytically competent manner. When isotope partitioning 
experiments were done with labeled ATP, in the absence of L- 
aspartate or L-glutamine (Table 2.2), 50% of the E-ATP* 
complex was trapped as AMP*. This shows that the E-ATP* 
complex is formed in a catalytically competent manner, 
further suggesting that ATP binds free enzyme first. When 
isotope partitioning experiments were done with L-aspartate 
in addition to the ATP* in the pulse solution, E-ATP*-Asp was 
trapped as AMP*. These data suggested that E-ATP*-Asp 
complex was formed in a catalytically competent manner. The 
produced AMP* was not stoichiometric with the amount of 
enzyme present suggesting that ATP hydrolysis was occurring 
prior to the addition of chase solution. The hydrolysis of 
ATP was found to be L-aspartate dependent. This partial 
reaction, although very slow (1/100 of the overall rate) , 
provides a direct evidence for the mechanism of E. coli ASB, 
in which the hydrolysis requires no nitrogen source. 



160 



The data presented indicated that for the glutamine- 
dependent AS reaction, the mechanism is ordered, with ATP 
binding first to free enzyme. It was also quite clear from 
the data that the binding and hydrolysis of L-glutamine is 
not required prior to the addition of ATP and L-aspartate, or 
no labeled L-asparagine would have been trapped in the 
absence of L-glutamine. Having said this, it was surprising 
that when L-glutamine was included in the pulse, about 90% of 
E-ATP*-Gln was trapped as AMP*, which suggested that an E- 
ATP*-Gln complex is formed in a catalytically competent 
manner. No ATP hydrolysis was stimulated by addition of L- 
glutamine, therefore suggesting that the presence of L- 
glutamine stabilized ATP binding to the active site because 
the amount of the AMP* trapped was stoichiometric with the 
amount of enzyme present. 

In the case of ammonia- dependent AS reaction, very 
little L-Asp* was trapped as L-Asn* when NH3 was included in 
the pulse solution with radioactive L-aspartate. The ability 
of ASB to trap radioactive L-aspartate (70%) when ATP was 
included in the pulse solution, shows that an E-Asp*-ATP 
complex is formed in a catalytically competent manner, 
implying that ATP binds first. On the other hand, when 
isotope partitioning experiments were done with labeled ATP 
and NH3 in the pulse, 20% of the E-ATP*-NH3 was trapped as 
AMP*. These data suggested that the NH3 binding is not 
required prior to the binding of ATP and L-aspartate or that 
the level of AMP* or L-Asn* trapped should be close to the 



161 



amount of enzyme present. The data also suggest that the 
mechanism is ordered such that ATP binds first followed by L- 
aspartate binding. 

It is quite obvious from this set of data that the 
mechanism for both the glutamine- and ammonia-dependent 
reactions is the same with ATP binding first to the free 
enzyme and L-aspartate second. This is followed by the 
release of PPi. This was further supported by aspartate- 
dependent ATP hydrolysis that requires no nitrogen source. 
Our proposed mechanism for ASB is completely different from 
other proposed mechanisms for AS (Milman et al., 1980, Markin 
et al., 1981 and Hongo and Sato, 1985) in that the former 
ones rationalized the ability of AS to behave as a 
glutaminase. They all agreed that L-glutamine is the first 
substrate to bind, and L-glutamate is the first product 
released. 

it was important, therefore, to study the kinetic 
mechanism of ASB using steady state kinetic methods in order 
to obtain more information about the overall mechanism and to 
determine whether the difference in the data was associated 
with using a different enzyme or with the technique employed 
(Chapter 3). Initial velocity experiments including product 
inhibition studies were performed. The data were in agreement 
with a bi-uni-uni-bi ping-pong mechanism (mechanism A) , for 
the ammonia-dependent reaction of ASB (see Chapter 3). This 
was further supported with the data from isotope trapping 
experiments showing that the mechanism is ordered and not 



162 



random, such that ATP binds first followed by L-aspartate 
binding (see Chapter 3) . However, it was interesting and 
frustrating at the same time to note that for the glutamine- 
dependent AS reaction neither one of the mechanisms (A or B) 
could be ruled out. Alternative substrate studies helped to 
explain some of the discrepancies that existed between the 
data presented in the past and this work. The product 
inhibition studies, single and/or double, provided 
information as to where some of the products can be placed. 
We went further and examined other mechanisms, including 
Milman's, uni-uni-bi-ter ping-pong Theroll-Chance mechanism, 
none of which fit our data. It became quite clear that steady- 
state kinetics alone could not distinguish between mechanism 
A (bi-uni-uni-bi ping-pong) from mechanism B (uni-uni-bi-ter 
ping-pong) for the glutamine-depenent reaction. The results 
from the isotope trapping experiments, however, indicated 
that the glutamine-dependent reaction the mechanism was 
ordered, ATP being first and L-aspartate second. These 
results together clearly support an ordered bi-uni-uni-ter 
ping-pong mechanism for the glutamine-dependent reaction of 
E. coli ASB. 

The product inhibition studies did not allow us to rule 
out any of the proposed mechanisms. Yet, they provided 
information as to where some of the products can be placed in 
the scheme. The rate equation for the proposed mechanism 
(Scheme A) was derived assuming the products P, Q, R and S 
were present, and the predicted product inhibition patterns 



163 



were compared with experimental results. There are some 
disagreements between the predicted patterns and the 
experimental results. One explanation was the poor ability of 
AMP and L-glutamate to inhibit the reaction. Another 
possibility was the existence of an isomerization step 
following the release of the last product (Scheme C) . The 
results obtained from studies appeared to be compatible with 
an iso ping-pong mechanism. 

Another very important observation made in this work was 
that the glutaminase reaction was shown to be occurring at 
the same time as the synthetase reaction, and in fact 
increasing with increasing concentration of L-glutamine. Such 
uncoupling of reactions was also reported for AS from 
leukemia cells (Horowitz and Meister, 1972) . They suggested 
that such uncoupling could be associated with the 
modification of the enzyme during isolation, for which they 
offered no evidence. It is also possible that such uncoupling 
observed with ASB may have something to do with cellular 
regulatory mechanism of this enzyme. 

Based on all these observations we came up with a model 
for the glutamine-dependent AS reaction. According to our 
model the reaction mechanism is more complex, and a simple 
ordered bi-uni-uni-ter ping-pong mechanism would not be 
applicable to E. coli ASB enzyme. We have two different 
reactions occurring at the same time, asparagine synthetase 
and glutaminase. The non-stoichiometry observed between 
formation of L-glutamate and PPi supports this proposed 



164 



model. The results from substrate inhibition studies are also 
in agreement with the presence of another pathway, that is 
selected over synthetase when substrate concentration is 
high. The steady-state initial velocity rate equation for the 
proposed mechanism (Scheme D) in the absence of products was 
derived, and computer modeling was used to examine the 
proposed mechanism through the simulation. We were able to 
show the change of pattern from intersecting lines to 
parallel lines using this complex mechanism, further 
supporting that the proposed mechanism (Scheme D) is right. 

Studying the kinetic mechanism allowed us to draw 
certain conclusions about the order of the binding of the 
substrates and release of products. It is quite clear that L- 
glutamate release can occur prior to the formation of 
aspartyl-AMP. However, the critical question is whether this 
early release of L-glutamate is required for the synthetase 
reaction to occur. Our studies showed that (1) the kinetic 
mechanism for glutamine-dependent reactions is preferentially 
ordered, with ATP binding first followed by addition of L- 
aspartate, and that (2) the binding and hydrolysis of L- 
glutamine is not required prior to the formation of aspartyl- 
AMP. The kinetic mechanism of ASB is a complex mechanism with 
two reactions occurring at the same time. Therefore, this 
makes it difficult to rule out any of the proposed chemical 
mechanisms . 

Furthermore, our examination of Ki ' s suggests that the 
potent inhibitors of AS should be analogs of L-asparagine 



165 



which was found to be competitive with L-glutamine (Ki = 
0.015 mM) . These inhibitors should not inhibit nor be 
hydrolyzed by any other enzyme such as asparaginase or 
asparagine transaminase. In addition, examination of Km's 
suggests that the analogs of L-glutamine would be better 
inhibitors of AS than that of ATP or L-aspartate. It would be 
easier to compete off L-glutamine with a Km of 0.2 mM as 
compared to that of ATP or L-aspartate (Km =0.05 mM) 

The combined studies using steady state and isotope 
partitioning analysis of ASB demonstrated the importance of 
relying on both techniques to understand enzymatic reaction 
mechanisms. It can be seen that an incomplete picture can be 
obtained from the steady state kinetics alone leading to an 
incomplete picture for the reactions occurring at the active 
site. Both approaches, therefore, together provide a more 
complete view of the kinetic pathway. 



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5436 















BIOGRAPHICAL SKETCH 

I, Pouran H-Tari, was born June of 1960 in Tehran, Iran. 
I got married the summer of 1978 and came to the United 
States. I lived in Sacramento for 3.5 years. Then I moved to 
Fresno and graduated in 1983 with a BA degree in biology from 
California State University. I then moved to Utah where I 
completed my master's degree in biological science with an 
emphasis in plant pathology from Utah State University, in 
Logan. I then went to work for a company called Gull 
Laboratory in Salt Lake City. In August of 1988, I moved to 
Gainesville, Florida. For one year I worked in protein core, 
after which I joined the Department of Biochemistry and 
Molecular Biology. I joined Dr. Schuster's laboratory the 
summer of 1990. I gave birth to my daughter Maryam on May 9, 
1992. I will receive my Ph.D. summer of 1996, after which I 
will go back home, to Tehran. I would stay there several 
months to relax and spend time with my daughter and my 
family. 



172 






I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

i // 



\ 



M^W 



Sheldon M. Schuster, Chair 
Professor of Biochemistry 
and Molecular Biology 
I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 



vlid(sL 



Robert $. Cohen 
Associate Professor of 
Biochemistry and Molecular 
Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in jscope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




Nigel (5. Richards 
Assistant Professor of 
Chemistry 

I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




Brian D. Cain 
Associate Professor of 
Biochemistry and Molecular 
Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

Ben M. Dunn 

Professor of Biochemistry 
and Molecular Biology 

This dissertation was submitted to the Graduate Faculty 
of the College of Medicine and to the Graduate School and was 
accepted as partial fulfillment of the requirements for the 
degree of Doctor of Philosophy. 



August 1996 





A. A*/*.*— .£>»— 



)ean, College of Medicine 
Dean, Graduate School 






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